U.S. patent application number 09/866209 was filed with the patent office on 2002-11-28 for methods for the identification of antimicrobial compounds.
Invention is credited to Nakata, Mitsunori, Selitrennikoff, Claude P..
Application Number | 20020177182 09/866209 |
Document ID | / |
Family ID | 25347158 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177182 |
Kind Code |
A1 |
Selitrennikoff, Claude P. ;
et al. |
November 28, 2002 |
Methods for the identification of antimicrobial compounds
Abstract
The present invention relates to methods to assay
2-amino-2-deoxy-D-glucos- e-6-phosphate ketol-isomerase activity.
The present invention also relates to methods for drug screening to
identify compounds having antimicrobial activity, wherein the
compounds have the ability to inhibit the enzymatic activity of a
microbial ketol-isomerase. In other embodiments, methods are
provided for the identification of compounds that selectively
inhibit microbial ketol-isomerase activity compared to the
ketol-isomerase activity of the subject being treated for an
infection.
Inventors: |
Selitrennikoff, Claude P.;
(Evergreen, CO) ; Nakata, Mitsunori; (Denver,
CO) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
101 HOWARD STREET
SUITE 350
SAN FRANCISCO
CA
94105
US
|
Family ID: |
25347158 |
Appl. No.: |
09/866209 |
Filed: |
May 25, 2001 |
Current U.S.
Class: |
435/25 ;
435/34 |
Current CPC
Class: |
C12Q 1/32 20130101; C12Q
1/533 20130101; C12Q 1/18 20130101 |
Class at
Publication: |
435/25 ;
435/34 |
International
Class: |
C12Q 001/26; C12Q
001/04 |
Claims
1. A method for the detection of glutamate in an experimental
sample, comprising the steps of: a) providing: i) an experimental
sample, ii) glutamate dehydrogenase, iii) nicotinamide adenine
dinucleotide, iv) nitro blue tetrazolium chloride, and v) phenazine
methosulfate; and b) combining said experimental sample, said
glutamate dehydrogenase activity, said nicotinamide adenine
dinucleotide, said nitro blue tetrazolium chloride, and said
phenazine methosulfate under conditions in which said nitro blue
tetrazolium chloride is reduced to yield a chromogenic product, and
wherein production of said chromogenic product is indicative of
glutamate in said experimental sample.
2. The method of claim 1, further wherein the concentration of
glutamate in said experimental sample is quantitated, providing at
least two control samples containing known concentrations of
glutamate, wherein the quantity of said chromogenic product formed
in step b) is proportional to the concentration of glutamate in
said samples, and the concentration of glutamate in the
experimental sample is determined by constructing a standard curve
using said control samples.
3. A method for the detection of ketol-isomerase activity in a
sample, comprising the sequential steps of: a) providing: i) a
sample, ii) fructose-6-phosphate, iii) glutamine, iv) glutamate
dehydrogenase, v) nicotinamide adenine dinucleotide, vi) nitro blue
tetrazolium chloride, and vii) phenazine methosulfate; b) combining
said sample, said fructose-6-phosphate, and said glutamine under
conditions to yield a reaction product comprising
glucosamine-6-phosphate and glutamate; c) inactivating said
ketol-isomerase activity; and d) combining said reaction product
with said glutamate dehydrogenase activity, said nicotinamide
adenine dinucleotide, said nitro blue tetrazolium chloride, and
said phenazine methosulfate under conditions wherein said nitro
blue tetrazolium chloride is reduced to yield a chromogenic
product, wherein production of said chromogenic product is
indicative of ketol-isomerase activity in said sample.
4. The method of claim 3, wherein said sample comprises a
ketol-isomerase selected from the group consisting of fungal
ketol-isomerases, bacterial ketol-isomerases, animal
ketol-isomerases and plant ketol-isomerases.
5. The method of claim 3, wherein said sample comprises a crude
cell lysate, selected from the group consisting of fungal cell
lysates, bacterial cell lysates, animal cell lysates and plant cell
lysates.
6. The method of claim 5, wherein said crude cell lysate is
selected from the group consisting of Aspergillus cell lysates,
Candida cell lysates, Cryptococcus cell lysates, Histoplasma cell
lysates, Pneumocystis cell lysates, Rhizopus cell lysates,
Saccharomyces cell lysates, Schizosaccharomyces cell lysates,
Escherichia cell lysates, Staphylococcus cell lysates and
Pseudomonas cell lysates.
7. The method of claim 3, wherein said sample comprises a purified
ketol-isomerase.
8. The method of claim 3, wherein said sample comprises a
recombinant ketol-isomerase.
9. The method of claim 8, wherein said recombinant ketol-isomerase
is a recombinant human ketol-isomerase.
10. The method of claim 3, wherein said inactivating step is
selected from the group consisting of boiling and heating to
70.degree. C.
11. The method of claim 3, further comprising a clarifying step
after the inactivating step, wherein said clarifying step is
selected from the group consisting of centrifugation, filtration
and a combination thereof.
12. A method for the identification of a compound having the
ability to inhibit a microbial ketol-isomerase activity, comprising
the sequential steps of: a) providing: i) microbial
ketol-isomerase, ii) fructose-6-phosphate, iii) glutamine, iv)
glutamate dehydrogenase, v) nicotinamide adenine dinucleotide, vi)
nitro blue tetrazolium chloride, vii) phenazine methosulfate, and
viii) a candidate compound; and b) preparing a first and a second
reaction mixture, wherein said first reaction mixture comprises
said microbial ketol-isomerase, said fructose-6-phosphate and said
glutamine, and wherein said second reaction mixture comprises said
microbial ketol-isomerase, said fructose-6-phosphate, said
glutamine and said candidate compound; c) exposing said first and
second reaction mixtures to conditions wherein said microbial
ketol-isomerase is capable of producing glucosamine-6-phosphate and
glutamate; d) inactivating said microbial ketol-isomerase; e)
combining said first and second reaction mixtures with said
glutamate dehydrogenase, said nicotinamide adenine dinucleotide,
said nitro blue tetrazolium chloride, and said phenazine
methosulfate under conditions wherein said nitro blue tetrazolium
chloride is capable of yielding a chromogenic product, wherein the
quantity of chromogenic product produced is proportional to
microbial ketol-isomerase activity; f) comparing ketol-isomerase
activities in said first and second reaction mixtures, and g)
identifying a candidate compound having the ability to inhibit said
microbial ketol-isomerase activity, wherein the microbial
ketol-isomerase activity of said first reaction mixture is greater
than the ketol-isomerase activity of said second reaction
mixture.
13. The method of claim 12, wherein said microbial ketol-isomerase
comprises a crude microbial cell lysate, selected from the group
consisting of fungal cell lysates and bacterial cell lysates.
14. The method of claim 13, wherein said crude microbial cell
lysate is selected from the group consisting of Aspergillus cell
lysates, Candida cell lysates, Cryptococcus cell lysates,
Histoplasma cell lysates, Pneumocystis cell lysates, Rhizopus cell
lysates, Saccharomyces cell lysates, Schizosaccharomyces cell
lysates, Escherichia cell lysates, Staphylococcus cell lysates and
Pseudomonas cell lysates.
15. The method of claim 12, wherein said inactivating step is
selected from the group consisting of boiling and heating to
70.degree. C.
16. The method of claim 12, further comprising a clarifying step
after the inactivating step, wherein said clarifying step is
selected from the group consisting of centrifugation, filtration
and a combination thereof.
17. The method of claim 12, wherein said compound has the ability
to inhibit a microbial ketol-isomerase activity, and further
testing said compound for antimicrobial activity using a testing
means.
18. The method of claim 17, wherein said testing means comprises at
least one method selected from the group consisting of agar
diffusion assays, broth dilution assays, in vivo mouse candidosis
assays, and in vivo mouse aspergillosis assays.
19. The method of claim 12, wherein said compound preferentially
inhibits said microbial ketol-isomerase compared to a second
ketol-isomerase, and wherein said method further comprises the
steps of: h) providing a second ketol-isomerase selected from the
group consisting of plant ketol-isomerases and animal
ketol-isomerases; and i) preparing a third and a fourth reaction
mixture, wherein said third reaction mixture comprises said second
ketol-isomerase, said fructose-6-phosphate and said glutamine, and
wherein said fourth reaction mixture comprises said second
ketol-isomerase, said fructose-6-phosphate, said glutamine and said
candidate compound, j) exposing said third and fourth reaction
mixtures to conditions wherein said second ketol-isomerase is
capable of producing glucosamine-6-phosphate and glutamate, k)
inactivating said second ketol-isomerase, l) combining said third
and fourth reaction mixtures with said glutamate dehydrogenase
activity, nicotinamide adenine dinucleotide, nitro blue tetrazolium
chloride, and phenazine methosulfate under conditions wherein said
nitro blue tetrazolium chloride is capable of yielding a
chromogenic product, wherein production of said chromogenic product
is proportional to said second ketol-isomerase activity, m)
comparing said second ketol-isomerase activities in said third and
fourth reaction mixtures, and n) identifying a compound which
preferentially inhibits said microbial ketol-isomerase activity
compared to said second ketol-isomerase activity.
20. The method of claim 19, wherein said animal ketol-isomerases
are mammalian ketol-isomerases.
21. The method of claim 20, wherein said mammalian ketol-isomerases
are selected from the group consisting of rat ketol-isomerases and
human ketol-isomerases.
22. The method of claim 19, wherein said second ketol-isomerase
comprises a cell lysate.
23. The method of claim 19, wherein said second ketol-isomerase is
purified.
24. The method of claim 19, wherein said second ketol-isomerase is
a recombinant ketol-isomerase.
25. The method of claim 24, wherein said recombinant
ketol-isomerase is a recombinant human ketol-isomerase.
26. The method of claim 19, wherein said inactivating step is
selected from the group consisting of boiling and heating to
70.degree. C.
27. The method of claim 19, further comprising a clarifying step
after the inactivating step, wherein said clarifying step is
selected from the group consisting of centrifugation, filtration
and a combination thereof.
28. The method of claim 19, wherein said compound preferentially
inhibits said microbial ketol-isomerase activity compared to a
second ketol-isomerase activity, and further testing said compound
for antimicrobial activity using a testing means.
29. The method of claim 28, wherein said testing means comprises at
least one method selected from the group consisting of agar
diffusion assays, broth dilution assays, in vivo mouse candidosis
assays, and in vivo mouse aspergillosis assays.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the assay of
2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase activity. The
present invention also relates to methods for drug screening to
identify compounds having antimicrobial activity, wherein the
compounds have the ability to inhibit the enzymatic activity of
microbial ketol-isomerase.
BACKGROUND
[0002] Microbial infections (e.g., infections by fungal or
bacterial species) account for significant morbidity and mortality
throughout the world. Although significant resources have been
dedicated to identifying compounds having antimicrobial properties,
microbial infections continue to present a significant human health
risk in both developed and undeveloped countries.
[0003] Bacterial Infections and Drug Resistance
[0004] The development of antibacterial agents, starting with the
identification of penicillin in the 1920s, has played a vital role
in the treatment of human infectious diseases. However, the recent
emergence of pathogenic microorganisms that are resistant to known
antimicrobial compounds is cause for great concern. This situation
has resulted from prolonged, worldwide use of antimicrobial
compounds, with the unfortunate effect of selection of resistant
microorganisms. For example, penicillin resistance has become
increasingly widespread in the bacterial species that were
previously susceptible to the drug. Some microorganisms produce
.beta.-lactamase, an enzyme which destroys the antimicrobial agent
itself, while some microorganisms have undergone genetic changes
resulting in alterations in the cell receptor protein to which the
penicillin binds (i.e., penicillin-binding proteins; PBPs; Jawetz,
Melnick & Adelberg's Medical Microbiology, 19th ed, Appleton
& Lange, Norwalk, Conn. [1991], p. 150), such that the drugs
will no longer effectively bind to the receptors. Still other
microorganisms have evolved mechanisms that prevent lysis of the
organism after the drug has bound to the cell. In this latter
scenario, the drug inhibits the growth of the organism, but the
organism is not killed, and relapse of disease occurs following
discontinuation of treatment.
[0005] One well-documented example highlighting the problem of drug
resistance is the development of penicillin resistance in the
pathogenic bacterium Streptococcus pneumoniae. Initially, the
introduction of penicillin to treat S. pneumoniae resulted in a
significant decrease in the mortality due to this organism.
However, S. pneumoniae infection remains of great concern, as it is
one of the organisms most frequently associated with invasive
infections; it is the most common cause of bacterial pneumonia and
otitis media, as well as the second most common cause of bacterial
meningitis, and the third most common isolate from blood cultures
(Sessegolo et al., J. Clin. Microbiol., 32:906-911 [1994]).
[0006] The first report of pneumococci with decreased
susceptibilities to penicillins occurred in 1967 in Australia.
Since this initial report, additional strains with decreased
susceptibilities have been reported worldwide. Additionally,
bacterial resistance to alternative antibacterial compounds, such
as chloramphenicol, erythromycin, tetracycline, clindamycin,
rifampin, and sulfamethoxazole-trimethoprim has also been reported,
often in conjunction with penicillin resistance.
Multiple-antimicrobial resistance in pneumococci was first reported
in 1977 in South Africa. Since this initial report, multi-drug
resistant strains have been reported in several other countries,
including Spain, Italy, France, Belgium, Hungary, Pakistan,
Czechoslovakia, Canada, the United Kingdom, and the United States
(Sessegolo et al., J. Clin. Microbiol., 32:906-911 [1994]).
[0007] A second example illustrating the evolution of a multiple
drug-resistant organism is Nisseria gonorrhoeae, the causative
agent of gonorrhea. Prior to the 1930s, treatment for this disease
was largely ineffective. In the late 1930s, sulfonamide
antibacterials were found to be effective in treating gonorrhea. A
few years thereafter, sulfonamide-resistant strains of N.
gonorrhoeae were identified. Fortunately, by this time, penicillin
was available and found to be effective against these infections.
However, by the 1970's, many isolates of N. gonorrhoeae were found
to be penicillin-resistant, requiring the use of additional,
alternative drugs such as spectinomycin. It can be expected that
this trend will continue, with the development of strains that are
resistant to sulfonamides, penicillin, spectinomycin, and other
antimicrobials.
[0008] Multiple drug resistance has been reported in a large number
of clinically significant bacterial species, including Escherichia
coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Haemophilus
influenzae, Neisseriae gonorrhoeae, Staphylococcus aureus,
Staphylococcus hemolyticus, and Streptococcus pneumoniae. Many of
these organisms are isolated from hospital environments. The
development of multi-drug-resistance of nosocomial
(hospital-acquired) and community-acquired pathogens to
antimicrobial agents is a significant public health concern with
both clinical and economic consequences.
[0009] Alarmingly, in the past few years, a handful of organisms
resistant to all known antimicrobial agents has emerged (Tenover et
al., Am. J. Med. Sci., 311:9-16 [1996]). Though such organisms are
rare, the existence of conditions favoring the development and
spread of these organisms forecasts the continued emergence of
multi-drug resistance. This problem is further exacerbated by the
scarcity of new classes of antimicrobial agents, since many
pharmaceutical manufacturers have abandoned the discovery of
antimicrobial drugs in favor of more profitable products.
[0010] Fungal Infections and Drug Resistance
[0011] Fungal organisms have become increasingly significant
pathogens in immunocompromised patients, especially those who
because of cancer, organ transplantation, chemotherapy, pregnancy,
age, diabetes, complications following extensive surgery, and
various immune system dysfunctions, are at risk of experiencing
life-threatening diseases caused by microorganisms which do not
ordinarily pose a threat to normal, immunocompetent people. Other
risk factors for deeply invasive fungal infections include
protracted treatment using broad spectrum antimicrobials,
corticosteroids, and vascular catheters.
[0012] Indeed, immunocompromised patients provide a significant
challenge to modem health care delivery. For example, fungal
infections have become one of the leading factors contributing to
morbidity and mortality in cancer patients, and fungi account for
4-12% of nosocomial pathogens in leukemia patients (Anaissie, Clin.
Infect. Dis., 14[Suppl.1]:S43 [1992]). The incidence of nosocomial
bloodstream infections with fungi such as Candida spp.
("candidemia") has increased in recent years and has been reported
to account for 5.6% of all primary bloodstream infections. There
are an estimated 200,000 patients/year who acquire nosocomial
fungal infections, with bloodstream infections having a mean
mortality rate of 55% (See e.g., Beck-Sague et al., J. Infect.
Dis., 167:1247 [1993]; and the Centers for Disease Control website
at www.cdc.gov/ncidod/publication- s/brochures/hip.html). Fungal
infections in non-humans, such as livestock and agricultural
products, is also of significant health and economic concern. The
most common fungal pathogens in humans are the opportunistic yeast,
Candida albicans and the filamentous mold, Aspergillus fumigatus
(See, Bow, Br. J. Haematol., 101:1 [1998]; and Warnock, J.
Antimicrob. Chemother., 41:95 [1998]).
[0013] C. albicans is the most common fungal pathogen in humans,
with other Candida species becoming increasingly important in
fungal disease in humans and other animals (See, Walsh and Dixon,
"Spectrum of Mycoses," in Baron [ed.], Medical Microbiology, 4th
ed, University of Texas Medical Branch, Galveston, Tex. [1996], pp.
919-925). Approximately 200 Candida species are known, with
approximately seven of those species isolated with significant
frequency from human specimens (See, Warren and Hazen, Ch. 95, pp.
1184-1199, "Candida, Cryptococcus, and Other Yeasts of Medical
Importance," in Murray et al., [eds], Manual of Clinical
Microbiology, 7th ed., ASM Press, Washington, D.C. [1999]; and
Mitchell, in Zinsser Microbiology, Joklik et al., [eds], Appleton,
Century-Crofts, Norwalk, Conn., pp. 1183-1190 [1984]).
[0014] The clinical manifestations of Candida infections and
disease are many and varied, as Candida species are known to invade
most organ systems of the body. Superficial candidiasis may involve
the epidermal and mucosal surfaces (e.g., the oral cavity, pharynx,
esophagus, intestines, urinary bladder, and vagina). In deep
candidiasis, the gastrointestinal tract and intravascular catheters
are the two major portals of entry, with the kidneys, liver,
spleen, brain, eyes, heart, and other tissues being the major sites
involved.
[0015] The major difficulties in treating Candida infections are
encountered in cases of systemic disease. Chronic mucocutaneous,
pulmonary candidiasis, endocarditis, and fungemia must be diagnosed
early in order to avoid fatality. The incidence of candidiasis in
certain patient populations is striking. Up to 30% of leukemia
patients acquire systemic candidiasis (Anaissie, Clin. Infect.
Dis., 14[Suppl.1]:S43 [1992]). This is of great significance, as
some reports indicate that the fatality rate for disseminated
candidiasis in cancer patients is as high as 80% (Meunier, et al.,
Clin. Infect. Dis., 14[Suppl. 1]:S120 [1992]).
[0016] Aspergillus species are the second most common isolate,
after Candida species, in patients with positive fungal cultures
(See, Sigler and Kennedy, Ch. 97, "Aspergillus, Fusarium, and Other
Opportunistic Moniliaceous Fungi," in Murray et al., (eds), Manual
of Clinical Microbiology, 7th ed., ASM Press, Washington, D.C.
[1999], pp. 1213-1241; and Goodwin et al, J. Med. Vet. Mycol.,
30:153 [1992]). A large number of species of the genus Aspergillus
have clinical relevance, although A. fumigatus, A. niger and A.
flavus are most commonly isolated. Of these isolates, A. fumigatus
is the most common human pathogen. Three main types of disease have
been associated with A. fumigatus, namely allergic asthma,
aspergilloma, and invasive aspergillosis (See e.g., Lortholary et
al., Amer. J. Med., 95:177-187 [1993]).
[0017] Allergic pulmonary asthma due to A. fumigatus exposure
affects an estimated 50,000 individuals in the U.S. Aspergillomas
are formed when fungal spores germinate in situ in tissue such as
the lungs and form fungus balls. There is typically no invasion of
underlying tissues, and in most cases treatment involves the simple
surgical removal of the aspergilloma. However, invasive
aspergillosis involves the invasion of host tissues, and is most
commonly observed in patients with predisposing conditions (e.g.,
immunosuppressive drugs, neutropenia, chemotherapy, AIDS).
Transplant (e.g., bone marrow or organ) and chemotherapy patients
are at the greatest risk for this form of aspergillosis (See e.g.,
Denning et al., New Eng. J. Med., 324:654-662 [1992]; and Miller et
al., Chest 105:37-44 [1994]). The prognosis for patients with
invasive aspergillosis is particularly grave, with mortality rates
greater than 50% (See e.g,, Polis et al., "Fungal Infections in
Patients with the Acquired Immunodeficiency Syndrome," in DeVita et
al. (eds), AIDS: Biology, Diagnosis Treatment and Prevention, 4th
ed., Lippincott-Raven, [1997]), due to the lack of a rapid
diagnostic method to confirm A. fumigatus infection, and the lack
of safe antifungal drugs.
[0018] The development of effective antifungal agents has lagged
behind that of antibacterial agents. Fungi, like humans, are
eukaryotic. Thus, most agents that have antimicrobial activity
towards fungi are also toxic to humans (i.e., due to non-selective
toxicity). Four general groups of antifungals have been developed;
these are the polyenes, the azoles, the allylamines/morpholines,
and the antimetabolites. Despite the identification of cell
membrane, cell wall, and microtubule targets for antifungal action,
antifungal development has been slow.
[0019] The polyene antifungals (e.g., amphotericin B and nystatin)
target the fungal cell membrane, which is similar to mammalian
plasma membranes, with the exception being that fungal plasma
membranes contain ergosterol, rather than cholesterol as the
principal sterol. The polyene amphotericin B remains the treatment
mainstay for life-threatening and other mycoses, including
candidiasis, cryptococcosis, aspergillosis, zygomycosis,
coccidioidomycosis, histoplasmosis, blastomycosis, and
paracoccidioidomycosis. Amphotericin B must be administered
intravenously and is associated with numerous, often serious side
effects, including phlebitis at the infusion site, fever, chills,
hypokalemia, anemia, nephrotoxicity and hypokalemia. Nystatin is
another broad-spectrum polyene antifungal. However, its toxicity to
humans prevents its widespread use. Currently, it is limited to
topical applications, where it is effective against yeasts,
including C. albicans.
[0020] The azole antifungals (e.g., fluconazole, itraconazole,
imidazole and ketoconazole) and the allylamine and morpholine
antifungals (e.g., naftifine and terbinafine) interfere with
ergosterol biosynthesis. Ketoconazole may be used to treat
histoplasmosis, blastomycosis, mucosal candidiasis and various
cutaneous mycoses (e.g., dermatophyte infections, pityriasis
versicolor, and cutaneous candidiasis). However, it is not useful
for treatment of aspergillosis or systemic yeast infections. Side
effects associated with use of the azoles are not as severe as
those associated with amphotericin B, although life-threatening
hepatic toxicity may result from long-term azole use. Other side
effects include nausea, vomiting, and drug interactions with such
compounds as cyclosporin, antihistamines, anticoagulants,
anti-seizure and oral hypoglycemic medications.
[0021] The few antimetabolite antifungals identified have not found
widespread use. The most commonly used antifungal is
5-fluorocytosine, a fluorinated analog of cytosine. However, as
with other antimetabolites, drug resistant fungal strains have
emerged, and 5-fluorocytosine is seldom used alone. Nonetheless, in
combination with amphotericin B, it remains the treatment of choice
for cryptococcal meningitis, and is effective against some diseases
caused by dematiaceous fungi.
[0022] Griseofulvin, an antifungal compound produced by Penicillium
griseofulvin, acts by targeting microtubule-associated proteins.
Griseofulvin is active against most dermatophytes, and is commonly
used to treat dermatophytic infections. Potassium iodide is another
compound that is used as an antifungal to enhance transepidermal
elimination of fungal organisms in cases of cutaneous and
lymphocutaneous sporotrichosis, although it is not effective
against Sporothrix schenckii in vitro.
[0023] As in bacteria, drug-resistant strains of fungal pathogens
have also been reported. This drug resistance can take various
forms, such as primary resistance, where the susceptibility
profiles for the species are characteristic, inherent, and rarely
change in response to drug exposure, or the resistance can be
secondary (i.e., acquired). Some of the molecular and cellular
mechanisms by which fungal organisms acquire resistance are known
(White, ASM News 63:427-433 [1997]; and White et al., Clin.
Microbiol. Rev., 11:382-402 [1998]).
[0024] Significantly, fungal resistance to amphotericin B has been
reported for various opportunistic fungi, including
Pseudallescheria boydii, Fusarium, Trichosporon, and some C.
lusitaniae and C. guilliermondii isolates (See, Dixon and Walsh,
"Antifungal Agents," in Baron (ed.), Medical Microbiology, 4th ed.,
University of Texas Medical Branch, Galveston, Tex. [1996], pp.
926-932). In addition, the emergence of azole-resistant fungal
strains has raised concerns regarding use of the azole compounds,
especially fluconazole, as a front-line treatment regime (Boschman
et al., Antimicrob. Agents Chemother., 42:734 [1998]; Graybill,
Clin. Infect. Dis., 22(Suppl.2):S166 [1996]; White, ASM News
63:427-433 [1997]; and White et al., Clin. Microbiol. Rev.,
11:382-402 [1998]).
[0025] In view of the development of resistance, as well as the
relative lack of variety available in the selection of antifungals,
there remains a need for the development of compounds useful for
treatment of fungal diseases.
[0026] Selective Toxicity
[0027] The principle of selective toxicity is fundamental to the
development of successful antimicrobial agents. That is to say, an
antimicrobial compound, while toxic to the microorganism, ideally
is not toxic to the subject receiving the antimicrobial compound.
Selective toxicity is often a reflection of differences between the
microorganism and host physiologies.
[0028] One approach to achieving selective toxicity is to identify
a compound that is able to inhibit an essential enzyme in the
microorganism, but due to differences in enzyme structure or
function, that same antimicrobial compound does not affect the
homologous enzyme in the host. Alternatively, an antimicrobial
compound can inhibit a biochemical event that is essential to the
microorganism, but that biochemical process may not be present or
be essential to the host.
[0029] For example, penicillin, like all .beta.-lactam
antibacterial drugs, is a compound that selectively inhibits
bacterial cell wall synthesis. The initial step in the mechanism of
action of these .beta.-lactam drugs involves the binding of the
drug to cell receptors known as "penicillin-binding proteins"
(PBPs) (Jawetz, Melnick & Adelberg's Medical Microbiology, 19th
ed, Appleton & Lange, Norwalk, Conn. [1991], p. 150). After
binding to the PBP, the drug inhibits the synthesis of
peptidoglycan, an essential component in the bacterial cell wall.
This results in the eventual triggering of an autolytic cascade
leading to cell lysis. Furthermore, these .beta.-lactam drugs are
remarkably non-toxic to humans and other animals, due to the
absence of peptidoglycans in animal cells.
[0030] Chitin Biosynthesis
[0031] The fungal cell wall is essential for the viability of the
organism, and is a rigid, stratified structure consisting of
chitinous microfibrils and polysaccharides, among other components.
The cell wall provides support and shape to the cell, and prevents
osmotic lysis of the cell. Indeed, even a small lesion within the
cell wall can lead to the extrusion of cytoplasm due to the
positive intracellular pressure (See, Cole, "Basic Biology of
Fungi, in Baron (ed.), Medical Microbiology, 4th ed., University of
Texas Medical Branch, Galveston, Tex. [1996], pp. 903-911). The
yeast form of the C. albicans cell wall contains approximately
30-60% glucan, 25-50% mannan (mannoprotein), 1-2% chitin, 2-14%
lipid, and 5-15% protein (McGinnis and Tyring, "Introduction to
Mycology," in Baron (ed.), Medical Microbiology, 4th ed.,
University of Texas Medical Branch, Galveston, Tex. [1996], pp.
893-902). The chitin within the fungal cell wall is a (.beta.
1-4)-linked polymer of N-acetyl glucosamine (GlcNAc) polymerized by
chitin synthase at the plasma membrane (See, FIG. 1).
[0032] Chitin, although a minor component of yeast and filamentous
fungal cell walls, is essential for cell viability and
mother-daughter cell separation. Chitin biosynthesis, which
requires uridine diphosphate-N-acetyl glucosamine (UDP-GlcNAc), is
complex and is catalysed by at least three gene products in S.
cerevisiae, and perhaps as many as six gene products in certain
filamentous molds (Bulawa, Ann. Rev. Microbiol., 47:505-534 [1993];
and Mellado et al., Mol. Genet., 246:353-359 [1995]). The three
yeast genes, csI, csII and csIII, each have homologues in C.
albicans and each performs a different intracellular function.
[0033] UDP-GlcNAc is the substrate for chitin synthase. Normal
levels of UDP-GlcNAc are required for chitin biosynthesis and
subsequent cell wall assembly and growth (Katz and Rosenberger,
Biochim. Biophys. Acta., 208:452-460 [1970]). The pathway for the
synthesis of UDP-GlcNAc, known as the Leloir pathway (Leloir and
Cardini, Biochim. Biophys. Acta., 12:15-22 [1953]) is shown in FIG.
1. In this Figure, chitin synthase activity is shown for context,
but is not considered part of the Leloir pathway. The first
pathway-specific enzyme is 2-amino-2-deoxy-D-glucose-6- -phosphate
ketol-isomerase (known simply as ketol-isomerase; E.C. 5.3.1.19 or
E.C. 2.6.1.16). The ketol-isomerase is an amino transferase that
forms glucosamine-6-phosphate (GlcN-6-P) and glutamate from
fructose-6-phosphate and glutamine (Selitrennikoff and Sonneborn,
Develop. Biol., 54:37-51 [1976]). The second enzyme in the pathway
is aminodeoxyglucosephosphate acetyltransferase (E.C. 2.4.1.4),
which converts S-acetyl CoA and GlcN-6-P to CoA and
N-acetylglucosamine-6-phosp- hate (GlcNAc-6-P) (Selitrennikoff and
Sonneborn, Develop. Biol., 54:37-51 [1976]). The third enzyme is
acetylaminodeoxyglucose phosphomutase (also known as
GlcNAc-phosphomutase; E.C. 2.7.5.2) which converts GlcNAc-6-P to
GlcNAc-1-phosphate, employing Glc-1,6-phosphate as a co-factor. The
most downstream enzyme is UTP:acetylaminodeoxyglucose-1-phosphate
uridylyl transferase (E.C. 2.7.7.23), which converts UTP and
GlcNAc-1-phosphate to UDP-GlcNAc and pyrophosphate (PP.sub.i)
(Selitrennikoff and Sonneborn, Biochim. Biophys. Acta., 451:408-416
[1976]; and Etchebehere et al., J. Bacteriol., 175:5022-5027
[1993]).
[0034] Chitin synthase has been a target for the identification of
antifungal compounds for over 30 years, yet only two classes of
compounds that target this enzyme have been identified. These are
the competitive substrate inhibitors, namely the polyoxins and the
nikkomycins. Each of these enzymatic inhibitors resembles the
structure of the substrate, UDP-GlcNAc, and has inhibition
constants (K.sub.i) in the .mu.M range (Decker et al., J. Gen.
Microbiol., 137:1805-1813 [1991]). Unfortunately, nikkomycin shows
rapid degradation in biological fluids in rat, mouse and rabbit
model systems (Tokumura and Horie, Biol. Pharm. Bull., 20:577-580
[1977]).
[0035] Thus, there remains a need to identify new antimicrobial
compounds, and more specifically, there is a need to identify and
develop novel classes of antimicrobial compounds that have not been
previously developed as antimicrobials, for the purpose of
circumventing preexisting drug resistance and provides efficacious
compounds to combat microbial disease. Indeed, there remains a need
to identify and develop new classes of antimicrobial compounds that
are effective against multiple-drug resistant organisms. In
addition, there is a need to identify and develop antimicrobial
compounds that demonstrate selective toxicity towards
microorganisms, but are not toxic, or have minimal (i.e.,
tolerable) toxicity, to animal hosts (e.g., humans).
SUMMARY OF THE INVENTION
[0036] The present invention relates to methods to identify
compounds having antimicrobial activity. These methods utilize a
biochemical screen to identify antimicrobial compounds by testing
the ability of the compound to inhibit the activity of microbial
2-amino-2-deoxy-D-glucose-6- -phosphate ketol-isomerase
(ketol-isomerase).
[0037] The present invention also relates to methods to identify
compounds having antimicrobial activity that are likely to have no
toxic effects, or minimal toxic effects, on the subject being
treated for the microbial infection. These methods utilize a
biochemical screen for ketol-isomerase activity using microbial
ketol-isomerase enzymes, and also use ketol-isomerase enzymes from
subjects that are to be treated for microbial infections to test
whether the candidate compound inhibits the subjects' (i.e., the
hosts') ketol-isomerase activity. Those compounds which inhibit
microbial ketol-isomerase activity, but do not significantly
inhibit the subjects' ketol-isomerase activity (i.e.,
preferentially inhibits the microbial ketol-isomerase) are
candidates for development as antimicrobial drugs that have no or
minimal toxicity to the subject being treated.
[0038] In one embodiment, the present invention provides methods
for the detection of glutamate in an experimental sample, where the
methods comprise the steps of providing an experimental sample,
glutamate dehydrogenase, nicotinamide adenine dinucleotide, nitro
blue tetrazolium chloride, and phenazine methosulfate; and
combining these reagents with the sample under conditions where the
nitro blue tetrazolium chloride is reduced to yield a chromogenic
product, and the quantity of the chromogenic product produced is
proportional to the amount of glutamate in the experimental sample.
In a related embodiment, the present invention provides a method
for quantitating the concentration of glutamate in the sample,
where control samples containing known concentrations of glutamate
are analyzed in parallel to the experimental sample, a standard
curve is constructed using the control samples, and the glutamate
concentration of the experimental sample is read from the standard
curve.
[0039] In other embodiments, the present invention provides methods
for the detection of ketol-isomerase activity in a sample, where
the sample, fructose-6-phosphate, and glutamine are combined to
produce glucosamine-6-phosphate and glutamate, the reaction is
stopped, the glutamate is combined with glutamate dehydrogenase,
nicotinamide adenine dinucleotide, nitro blue tetrazolium chloride,
and phenazine methosulfate, where the nitro blue tetrazolium
chloride is reduced to yield a chromogenic product, and the
generation of the chromogenic product is proportional to the
ketol-isomerase activity in the original sample.
[0040] In alternative embodiments of these methods, the sample
being tested comprises fungal ketol-isomerases, bacterial
ketol-isomerases, animal ketol-isomerases or plant
ketol-isomerases. In other embodiments, the sample comprises a
fungal cell lysate, a bacterial cell lysate, an animal cell lysate
or a plant cell lysate. In alternative embodiments, the fungal cell
lysate is from Aspergillus spp., Candida spp., Cryptococcus spp.,
Histoplasma spp., Pneumocystis spp., Rhizopus spp., Saccharomyces
spp. (e.g., S. cerevisiae) or Schizosaccharomyces spp. (e.g., S.
pombe). In other alternative embodiments, the bacterial cell lysate
is selected from lysates of Escherichia spp. (e.g., E. coli),
Staphylococcus spp. and Pseudomonas spp. In still other embodiments
of these methods, the sample is a purified ketol-isomerase, while
in other embodiments the sample is a recombinant ketol-isomerase.
In one embodiment, the recombinant ketol-isomerase is a recombinant
human ketol-isomerase.
[0041] In some embodiments of these methods for the detection of
ketol-isomerase activity in a sample, the inactivation step for
stopping the ketol-isomerase reaction which produces glutamate is
accomplished by boiling, while in other embodiments, inactivation
is by heating to 70.degree. C. Furthermore, in other embodiments,
after the inactivation step, the reaction can be clarified using
either centrifugation, filtration or a combination of both
techniques.
[0042] In one embodiment, the present invention provides a method
for the identification of a compound having the ability to inhibit
a microbial ketol-isomerase activity. In this embodiment, two
reactions are prepared, where the first reaction comprises a
microbial ketol-isomerase, fructose-6-phosphate and glutamine, and
where the second reaction comprises a microbial ketol-isomerase,
fructose-6-phosphate, glutamine and a candidate compound. In this
embodiment, the reactions are run under conditions where the
microbial ketol-isomerase produces glucosamine-6-phosphate and
glutamate, the microbial ketol-isomerase reactions are then
inactivated, the reaction mixtures are combined with glutamate
dehydrogenase, nicotinamide adenine dinucleotide, nitro blue
tetrazolium chloride, and phenazine methosulfate under conditions
where the nitro blue tetrazolium chloride yields a chromogenic
product, where the quantity of chromogenic product produced is
proportional to microbial ketol-isomerase activity, and finally,
comparing the ketol-isomerase activities in the first and second
reaction mixtures, where if the ketol-isomerase activity of the
first reaction mixture is greater than the ketol-isomerase activity
of the second reaction mixture, the candidate compound has the
ability to inhibit the microbial ketol-isomerase activity.
[0043] In alternative embodiments of these methods, the microbial
ketol-isomerase comprises a crude fungal cell lysate, while in
other embodiments, the microbial ketol-isomerase comprises a
bacterial cell lysate. In alterantive embodiments, the fungal cell
lysate is from Aspergillus spp., Candida spp., Cryptococcus spp.,
Histoplasma spp., Pneumocystis spp., Rhizopus spp., Saccharomyces
spp. (e.g., S. cerevisiae) or Schizosaccharomyces spp. (e.g., S.
pombe). In other alternative embodiments, the bacterial cell lysate
is selected from lysates of Escherichia spp., (e.g., E. coli),
Staphylococcus spp. and Pseudomonas spp.
[0044] In some embodiments of these methods for the detection of
compounds that can inhibit ketol-isomerase activity, the
inactivation step for stopping the ketol-isomerase reaction which
produces glutamate is accomplished by boiling, while in other
embodiments, inactivation is by heating to 70.degree. C.
Furthermore, in other embodiments, after the inactivation step, the
reaction can be clarified using either centrifugation, filtration
or a combination of both techniques.
[0045] In other embodiments of the present invention, a compound
with the ability to inhibit a microbial ketol-isomerase activity is
further tested for antimicrobial activity. In one embodiment, the
method for testing for antimicrobial activity is an agar diffusion
assay, while in another embodiment, the testing method is a broth
dilution assay, while in another embodiment, the testing method is
an in vivo mouse candidosis assay, and in yet another embodiment,
the testing method is an in vivo mouse aspergillosis assay.
[0046] In other embodiments, the present invention provides methods
for the identification of compounds that preferentially inhibit
microbial ketol-isomerase rather than another (i.e., a second)
ketol-isomerase. In this embodiment, third and fourth reaction
mixtures are prepared, where the third reaction mixture comprises
the other (i.e., second) ketol-isomerase, fructose-6-phosphate and
glutamine, and where the fourth reaction mixture comprises the
second ketol-isomerase, fructose-6-phosphate, glutamine and the
candidate compound previously shown to inhibit microbial
ketol-isomerase. In this embodiment, the third and fourth reaction
mixtures are exposed to conditions where the second ketol-isomerase
is capable of producing glucosamine-6-phosphate and glutamate,
followed by inactivating the second ketol-isomerase, combining the
third and fourth reaction mixtures with glutamate dehydrogenase
activity, nicotinamide adenine dinucleotide, nitro blue tetrazolium
chloride, and phenazine methosulfate under conditions where the
nitro blue tetrazolium chloride is capable of producing a
chromogenic product where the amount of chromogenic product
produced is proportional to the amount of ketol-isomerase activity,
and finally, comparing the microbial ketol-isomerase activities in
the first and second reaction tubes, and comparing the second
ketol-isomerase activities in the third and fourth reaction
mixtures, and identifying a compound which preferentially inhibits
the microbial ketol-isomerase activity compared to the second
ketol-isomerase activity.
[0047] In some embodiments of this invention, the second
ketol-isomerase is a plant ketol-isomerase, while in other
embodiments, the second ketol-isomerase is an animal
ketol-isomerase. In another embodiment, the animal ketol-isomerases
are mammalian ketol-isomerases. In still further embodiments, the
mammalian ketol-isomerases are rat ketol-isomerases, while in
alternative embodiments, the mammalian ketol-isomerases are human
ketol-isomerases.
[0048] In some embodiments, the second ketol-isomerase comprises a
cell lysate. In some embodiments, the second ketol-isomerase is
purified. In some embodiments, the second ketol-isomerase is a
recombinant ketol-isomerase. In other embodiments, the recombinant
ketol-isomerase is a recombinant human ketol-isomerase.
[0049] In some embodiments of these methods for the identification
of compounds that preferentially inhibit microbial ketol-isomerase
activity, the inactivation step for stopping the ketol-isomerase
reaction which produces glutamate is accomplished by boiling, while
in other embodiments, inactivation is by heating to 70.degree. C.
Furthermore, in other embodiments, after the inactivation step, the
reaction can be clarified using either centrifugation, filtration
or a combination of both techniques.
[0050] In other embodiments, where a compound preferentially
inhibits a microbial ketol-isomerase activity compared to a second
ketol-isomerase activity, that compound is further tested for
antimicrobial activity using a testing means. In some embodiments,
the means for testing antimicrobial activity is an agar diffusion
assay, while in another embodiment, the testing method is a broth
dilution assay, while in another embodiment, the testing method is
an in vivo mouse candidosis assay, and in yet another embodiment,
the testing method is an in vivo mouse aspergillosis assay.
DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 shows the Leloir enzymatic pathway for the synthesis
of the chitin precursor UDP-N-acetylglucosamine (UDP-GlcNAc). The
chitin synthase step is also indicated, although this step is not
formally part of the Leloir Pathway.
[0052] FIG. 2 shows the ketol-isomerase/glutamate
dehydrogenase/nitro BT assay reaction steps.
[0053] FIG. 3 shows a standardization plot for glutamate (Glu)
concentration using the nitro blue tetrazolium chloride (nitro BT)
assay, where the glutamate concentration ranges from 0 to 600
.mu.M.
[0054] FIG. 4 shows two plots of ketol-isomerase activity of a
crude Aspergillus fumigatus cell extract using two different assay
methods. One plot provides results of the Morgan-Elson assay to
determine the concentration of the ketol-isomerase reaction product
glucosamine-6-phosphate (GlnN-6-P; open triangles). The second plot
provides results of the nitro BT assay to determine the
concentration of the ketol-isomerase reaction product glutamate
(Glu; closed circles).
[0055] FIG. 5 provides a histogram plot showing the ability of
UDP-N-acetylglucosamine (UDP-GlcNAc) to inhibit the activity of
ketol-isomerase (feedback inhibition) from a crude A. fumigatus
cell extract, as measured by the nitro BT assay, where the
UDP-GlcNAc concentration ranges from 0 to 10 mM.
DEFINITIONS
[0056] To facilitate understanding of the invention, a number of
terms are defined and discussed below.
[0057] The terms "nucleic acid," "nucleic acid sequence,"
"nucleotide sequence," "oligonucleotide," "polynucleotide" or
"nucleic acid molecule" as used herein refer to an oligonucleotide
or polynucleotide, and fragments or portions thereof, and to DNA or
RNA of genomic or synthetic origin which can be single- or
double-stranded, and represent the sense or antisense strand.
Similarly, "amino acid sequence" as used herein refers to the
primary sequence of amino acids in a peptide, polypeptide or
protein.
[0058] The term "nucleotide" as used herein refers to any
nucleotide that comprises any of the known base analogs of DNA and
RNA.
[0059] As used herein, "recombinant nucleic acid," "recombinant
gene" "recombinant DNA molecule" or similar terms indicate that the
nucleotide sequence or arrangement of its parts is not a native
configuration, and has been manipulated by molecular biological
techniques. The term implies that the DNA molecule is comprised of
segments of DNA that have been artificially joined together.
Protocols and reagents to manipulate nucleic acids are common and
routine in the art (See e.g., Maniatis et al.(eds.), Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
N.Y., [1982]; Sambrook et al. (eds.), Molecular Cloning: A
Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor
Laboratory Press, N.Y., [1989]; and Ausubel et al. (eds.), Current
Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons,
Inc., New York [1994]).
[0060] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to bacterial enzymes, each of which cut
double-stranded DNA at or near a specific nucleotide sequence.
[0061] The term "homology," as it applies to nucleotide sequences,
refers to a degree of complementarity. It is intended that the term
encompass partial homology as well as complete homology (i.e., 100%
identity). A partially complementary sequence is one that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid, and is referred to using the
functional term "substantially homologous." The inhibition of
hybridization of the completely complementary sequence to the
target sequence can be examined using a hybridization assay
(Southern or Northern blot, solution hybridization and the like)
under conditions of low stringency. A substantially homologous
sequence or probe will compete for and inhibit the binding (i.e.,
the hybridization) of a completely homologous sequence to a target
sequence under conditions of low stringency. This is not to say
that conditions of low stringency are such that non-specific
binding is permitted; low stringency conditions require that the
binding of two sequences to one another be a specific (i.e.,
selective) interaction. The absence of non-specific binding can be
tested by the use of a second target which lacks even a partial
degree of complementarity (e.g., less than about 30% identity); in
the absence of non-specific binding, the probe will not hybridize
to the second non-complementary target.
[0062] The terms "peptide," "polypeptide" and "protein" all refer
to a primary sequence of amino acids that are joined by covalent
"peptide linkages." In general, a peptide consists of a few amino
acids, typically from 2-25 amino acids, and is shorter than a
protein. "Polypeptides" encompass both peptides or proteins. As
used herein, a recited "amino acid sequence" refers to an amino
acid sequence of a naturally occurring protein molecule, a protein
produced by recombinant molecular genetic techniques, or a
synthetic or naturally occurring peptide, and may refer to a
portion of a larger "peptide," "polypeptide" or "protein," and is
not meant to limit the amino acid sequence to the complete, native
amino acid sequence associated with the recited protein
molecule.
[0063] A "recombinant protein" or "recombinant polypeptide" refers
to a protein molecule that is expressed from a recombinant DNA
molecule. Use of these terms indicates that the primary amino acid
sequence, arrangement of its domains or nucleic acid elements which
control its expression are not native, and have been manipulated by
molecular biology techniques. As indicated above, techniques to
manipulate recombinant proteins are also common and routine in the
art.
[0064] The terms "exogenous" and "heterologous" are sometimes used
interchangeably with "recombinant." An "exogenous nucleic acid,"
"exogenous gene" and "exogenous protein" indicate a nucleic acid,
gene or protein, respectively, that has come from a source other
than its native source, and has been artificially supplied to the
biological system. In contrast, the terms "endogenous protein,"
"native protein," "endogenous gene," and "native gene" refer to a
protein or gene that is native to the biological system, species or
chromosome under study. A "native" or "endogenous" gene is a gene
that does not contain nucleic acid elements encoded by sources
other than the chromosome on which it is normally found in nature.
An endogenous gene or transcript is encoded by its natural
chromosomal locus, and not artificially supplied to the cell.
[0065] As used herein the term "portion" when in reference to a
protein (as in "a portion of a given protein") refers to fragments
of that protein. In some embodiments, the fragments range in size
from four amino acid residues to the entire amino acid sequence
minus one amino acid. In other embodiments, the "portion" is
further limited to only fragments of the full length protein that
retain biological activity. For example, a portion of a
ketol-isomerase protein is a fragment of a ketol-isomerase protein
that retains the ability to catalyze the synthesis of
glucosamine-6-phosphate and glutamate from fructose-6-phosphate and
glutamine. Functional portions of the ketol-isomerase protein find
use with the present invention.
[0066] The following definitions are the commonly accepted
definitions of the terms "identity," "similarity" and "homology."
Percent identity, as it applies to polypeptides, is a measure of
strict amino acid conservation. Percent similarity is a measure of
amino acid conservation which incorporates both strictly conserved
amino acids, as well as "conservative" amino acid substitutions,
where one amino acid is substituted for a different amino acid
having similar chemical properties (i.e., a "conservative"
substitution). In some embodiments, the term "homology" pertains to
either proteins or nucleic acids. Two proteins be described as
"homologous" or "non-homologous," but the degree of amino acid
conservation is quantitated by percent identity and percent
similarity. Nucleic acid conservation is measured by the strict
conservation of the bases adenine, thymine, guanine and cytosine in
the primary nucleotide sequence. When describing nucleic acid
conservation, conservation of the nucleic acid primary sequence is
sometimes expressed as percent homology. In the same nucleic acid,
one region may show a high percentage of nucleotide sequence
conservation, while a different region shows no or poor
conservation. It is not possible to infer nucleotide sequence
conservation from an amino acid similarity score. Indeed, it is
possible for two proteins to show domains that in one region are
homologous, while other regions of the same protein the domains are
non-homologous.
[0067] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated nucleic acid," "an isolated oligonucleotide,"
"isolated polynucleotide" or "isolated nucleotide sequence," refers
to a nucleic acid that is identified and separated from at least
one contaminant nucleic acid with which it is ordinarily associated
in its natural source. Isolated nucleic acid is present in a form
or setting that is different from the form or setting of that
nucleic acid found in nature. In contrast, non-isolated nucleic
acids are found in the state in which they exist in nature. For
example, a given DNA sequence (e.g., a gene) is found on the host
cell chromosome in proximity to neighboring genes; RNA sequences,
such as a specific mRNA sequence encoding a specific protein, are
found in the cell in a mixture with numerous other mRNAs that
encode a multitude of proteins. However, isolated nucleic acid
encoding a given polypeptide includes, by way of example, such
nucleic acid in cells ordinarily expressing the given protein where
the nucleic acid is in a chromosomal location different from that
of natural cells, or is otherwise flanked by a different nucleic
acid sequence than that found in nature. This isolated nucleic
acid, oligonucleotide, or polynucleotide is either single-stranded
or double-stranded. When an isolated nucleic acid, oligonucleotide
or polynucleotide is to be utilized to express a protein, the
oligonucleotide or polynucleotide will contain at a minimum the
sense or coding strand (i.e., the oligonucleotide or polynucleotide
is single-stranded). In other embodiments, the oligonucleotide or
polynucleotide contains both the sense and anti-sense strands
(i.e., the oligonucleotide or polynucleotide is
double-stranded).
[0068] As used herein, the terms "purified," "to purify" or
"enriched" refer to the removal of at least one contaminant from a
sample. As used herein, the term "substantially purified" refers to
molecules, either nucleic acids or amino acid sequences, that are
removed from their natural environment, "isolated" or "separated,"
and are largely free from other components with which they are
naturally associated. An "isolated nucleic acid" or "isolated
polypeptide" are therefore a substantially purified nucleic acid or
substantially purified polypeptide. For example, antibodies are
purified by removal of contaminating non-immunoglobulin proteins;
they are also purified by the removal of non-specific
immunoglobulin that does not bind to the target molecule. The
removal of non-immunoglobulin proteins and/or the removal of
immunoglobulins that do not bind to the target molecule results in
an increase in the percent of target-reactive immunoglobulins in
the sample. The removal of non-immunoglobulin proteins and/or the
removal of immunoglobulins that do not bind to the target molecule
results in an increase in the percent of target-reactive
immunoglobulins in the sample (i.e., "enrichment" of an antibody).
In another example, recombinant polypeptides are expressed in
bacterial host cells and the polypeptides are purified by the
removal of host cell proteins; the percent of recombinant
polypeptides relative to all polypeptides in the sample is thereby
increased.
[0069] Nucleic acid molecules (e.g., DNA or RNA) are said to have
"5' ends" and "3' ends" because mononucleotides are reacted to make
oligonucleotides or polynucleotides in a manner such that the 5'
phosphate of one mononucleotide pentose ring is attached to the 3'
oxygen of its neighbor in one direction via a phosphodiester
linkage. Therefore, an end of an oligonucleotides or
polynucleotide, referred to as the "5' end" if its 5' phosphate is
not linked to the 3' oxygen of a mononucleotide pentose ring and as
the "3' end" if its 3' oxygen is not linked to a 5' phosphate of a
subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide or
polynucleotide, also can be said to have 5' and 3' ends. In either
a linear or circular DNA molecule, discrete elements are referred
to as being "upstream" or 5' of the "downstream" or 3' elements.
This terminology reflects the fact that transcription proceeds in a
5' to 3' fashion along the DNA strand. The promoter and enhancer
elements that direct transcription of a linked gene are generally
located 5' or upstream of the coding region. However, in some
embodiments, enhancer elements exert their effect even when located
3' of the promoter element or the coding region. Transcription
termination and polyadenylation signals are located 3' or
downstream of the coding region.
[0070] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence comprised of parts, that when appropriately combined in
either a native or recombinant manner, provide some product or
function. In some embodiments, genes comprise coding sequences
necessary for the production of a polypeptide, while in other
embodiments, the genes do not comprise coding sequences necessary
for the production of a polypeptide. Examples of genes that do not
encode polypeptide sequences include ribosomal RNA genes (rRNA) and
transfer RNA (tRNA) genes. In preferred embodiments, genes encode a
polypeptide or any portion of a polypeptide within the gene's
"coding region" or "open reading frame." In some embodiments, the
polypeptide produced by the open reading frame of a gene displays
functional activity or properties of the full-length polypeptide
(e.g., enzymatic activity, ligand binding, signal transduction,
etc.), while in other embodiments, it does not.
[0071] In addition to the coding region of the nucleic acid, the
term "gene" also encompasses the transcribed nucleotide sequences
of the full-length mRNA adjacent to the 5' and 3' ends of the
coding region. These noncoding regions are variable in size, and
typically extend for distances up to or exceeding 1 kb on both the
5' and 3' ends of the coding region. The sequences that are located
5' and 3' of the coding region and are contained on the mRNA are
referred to as 5' and 3' untranslated sequences (5' UT and 3' UT).
Both the 5' and 3' UT may serve regulatory roles, including
translation initiation, post-transcriptional cleavage and
polyadenylation. The term "gene" encompasses mRNA, cDNA and genomic
forms of a gene.
[0072] In some embodiments, the genomic form or genomic clone of a
gene contains the sequences of the transcribed mRNA, as well as
other non-coding sequences which lie outside of the mRNA. The
regulatory regions which lie outside the mRNA transcription unit
are sometimes called "5' or 3' flanking sequences." A functional
genomic form of a gene must contain regulatory elements necessary
for the regulation of transcription. The term "promoter/enhancer
region" is usually used to describe this DNA region, typically but
not necessarily 5' of the site of transcription initiation,
sufficient to confer appropriate transcriptional regulation. Used
alone, the term "promoter" is sometimes used synonymously with
"promoter/enhancer." In some embodiments, the promoter is
constitutively active, or while in alternative embodiments, the
promoter is conditionally active (i.e., where transcription is
initiated only under certain physiological conditions or in the
presence of certain drugs). In some embodiments, the 3' flanking
region contains additional sequences which regulate transcription,
especially the termination of transcription. "Introns" or
"intervening regions" or "intervening sequences" are segments of a
gene which are contained in the primary transcript (i.e.,
hetero-nuclear RNA, or hnRNA), but are spliced out to yield the
processed mRNA form. In some embodiments, introns contain
transcriptional regulatory elements such as enhancers. The mRNA
produced from the genomic copy of a gene is translated in the
presence of ribosomes to yield the primary amino acid sequence of
the polypeptide.
[0073] As used herein, the term "regulatory element" refers to a
genetic element which controls some aspect of the expression of
nucleic acid sequences. For example, a promoter is a regulatory
element that enables the initiation of transcription of an operably
linked coding region. Other regulatory elements are splicing
signals, polyadenylation signals, termination signals, etc.
[0074] Transcriptional control signals in eukaryotes comprise
"promoter" and "enhancer" elements. Promoters and enhancers consist
of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (Maniatis et al.,
Science 236:1237 [1987]). Promoter and enhancer elements have been
isolated from a variety of eukaryotic sources including genes in
yeast, insect and mammalian cells, as well as viruses. Analogous
control elements (i.e., promoters and enhancers) are also found in
prokaryotes. The selection of a particular promoter and enhancer to
be operably linked in a recombinant gene depends on what cell type
is to be used to express the protein of interest. Some eukaryotic
promoters and enhancers have a broad host range while others are
functional only in a limited subset of cell types (for review see,
Voss et al., Trends Biochem. Sci., 11:287 [1986] and Maniatis et
al., Science 236:1237 [1987]). For example, the SV40 early gene
enhancer is very active in a wide variety of mammalian cell types
(Dijkema et al., EMBO J., 4:761 [1985]). Two other examples of
promoter/enhancer elements active in a broad range of mammalian
cell types are those from the human elongation factor 1.alpha. gene
(Uetsuki et al., J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene
91:217 [1990]; Mizushima and Nagata, Nuc. Acids. Res., 18:5322
[1990]), the long terminal repeats of the Rous sarcoma virus
(Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 [1982]), and
human cytomegalovirus (Boshart et al., Cell 41:521 [1985]). Some
promoter elements serve to direct gene expression in a
tissue-specific manner.
[0075] As used herein, the term "promoter/enhancer" denotes a
segment of DNA which contains sequences capable of providing both
promoter and enhancer functions (i.e., the functions provided by a
promoter element and an enhancer element). For example, the long
terminal repeats of retroviruses contain both promoter and enhancer
functions. In some embodiments, the promoter/enhancer is
"endogenous," while in other embodiments, the promoter/enhancer is
"exogenous," or "heterologous." An "endogenous" promoter/enhancer
is one which is naturally linked with a given gene in the genome.
An "exogenous" or "heterologous" promoter/enhancer is one placed in
juxtaposition to a gene by means of genetic manipulation (i.e.,
molecular biological techniques such as cloning and recombination)
such that transcription of the gene is controlled by the linked
promoter/enhancer.
[0076] The terms "in operable combination," "in operable order,"
"operably linked" and similar phrases when used in reference to
nucleic acid herein are used to refer to the linkage of nucleic
acid sequences in such a manner that a nucleic acid molecule
capable of directing the transcription of a given gene and/or the
synthesis of a desired protein molecule is produced. The term also
refers to the linkage of amino acid sequences in such a manner so
that a functional protein is produced.
[0077] As used herein, the terms "an oligonucleotide having a
nucleotide sequence encoding a gene," "polynucleotide having a
nucleotide sequence encoding a gene," and similar phrases are meant
to indicate a nucleic acid sequence comprising the coding region of
a gene (i.e., the nucleic acid sequence which encodes a gene
product). In some embodiments, the coding region is present in a
cDNA, while in other embodiments, the coding region is present in
genomic DNA or RNA form. When present in a DNA form, the
oligonucleotide, polynucleotide or nucleic acid is either
single-stranded (i.e., the sense strand or the antisense strand) or
double-stranded. In some embodiments, suitable control elements
such as enhancers/promoters, splice junctions, polyadenylation
signals, etc. are placed in close proximity to the coding region of
the gene if needed to permit proper initiation of transcription
and/or correct processing of the primary RNA transcript.
Alternatively, the coding region utilized in the expression vectors
of the present invention contains endogenous enhancers/promoters,
splice junctions, intervening sequences, polyadenylation signals,
etc. or a combination of both endogenous and exogenous control
elements.
[0078] As used herein, the terms "nucleic acid molecule encoding,"
"DNA sequence encoding," and "DNA encoding" and similar phrases
refer to the order or sequence of deoxyribonucleotides along a
strand of deoxyribonucleic acid encoding a particular polypeptide.
The order of the deoxyribonucleotides determines the order of the
amino acids in the polypeptide chain. The DNA sequence thus codes
for the amino acid sequence.
[0079] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (i.e., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
the mRNA. Gene expression regulation often occurs at many stages.
"Up-regulation" or "activation" refers to regulation that increases
the production of gene expression products (i.e., RNA or protein),
while "down-regulation" or "repression" refers to regulation that
decreases mRNA or protein production. Molecules (e.g.,
transcription factors) that are involved in up-regulation or
down-regulation are often called "activators" and "repressors,"
respectively.
[0080] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector." In some embodiments, a vector "backbone" comprises
those parts of the vector which mediate its maintenance and enable
its intended use (e.g., the vector backbone contains sequences
necessary for replication, genes imparting drug or antibiotic
resistance, a multiple cloning site, and possibly operably linked
promoter/enhancer elements which enable the expression of a cloned
nucleic acid). The cloned nucleic acid (e.g., such as a cDNA coding
sequence, or an amplified PCR product) is inserted into the vector
backbone using common molecular biology techniques. Vectors are
often derived from plasmids, bacteriophages, or plant or animal
viruses. A "cloning vector" or "shuttle vector" or "subcloning
vector" contain operably linked parts which facilitate subcloning
steps (e.g., a multiple cloning site containing multiple
restriction endonuclease sites). A "recombinant vector" indicates
that the nucleotide sequence or arrangement of its parts is not a
native configuration, and has been manipulated by molecular
biological techniques. The term implies that the vector is
comprised of segments of DNA that have been artificially
joined.
[0081] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
operably linked nucleic acid sequences necessary for the expression
of the operably linked coding sequence in a particular host
organism (e.g., a bacterial expression vector, a yeast expression
vector or a mammalian expression vector). Nucleic acid sequences
necessary for expression in prokaryotes typically include a
promoter, an operator (optional), and a ribosome binding site,
often along with other sequences. Eukaryotic cells utilize
promoters, enhancers, and termination and polyadenylation signals
and other sequences which are different from those used by
prokaryotes.
[0082] The term "transgene" as used herein refers to a foreign gene
that is placed into an organism by, for example, introducing the
foreign gene into newly fertilized eggs or early embryos. The term
"foreign gene" refers to any nucleic acid (e.g., gene sequence)
that is introduced into the genome of an animal by experimental
manipulations and in some embodiments, include gene sequences found
in that animal so long as the introduced gene does not reside in
the same location as does the naturally-occurring gene.
[0083] The terms "overexpression" and "overexpressing" and
grammatical equivalents are used in reference to levels of mRNA or
protein where the level of expression of the mRNA or protein is
higher than that typically observed in a given cell (prokaryotic or
eukaryotic) or in a tissue in a control or non-transgenic animal.
Levels of mRNA or protein are measured using any of a number of
techniques known to those skilled in the art. For example, in some
embodiments mRNA levels are assayed using methods such as Northern
blot analysis. Appropriate controls are included on the Northern
blot to control for differences in the amount of RNA loaded from
each tissue analyzed (e.g., the amount of 28S rRNA, an abundant RNA
transcript present at essentially the same amount in all tissues,
present in each sample is used as a means of normalizing or
standardizing the mRNA-specific signal observed on Northern blots).
The amount of mRNA present in the band corresponding in size to the
correctly spliced transgene RNA is quantified; other minor species
of RNA which hybridize to the transgene probe are not considered in
the quantification of the expression of the transgenic mRNA.
[0084] The term "transfection" as used herein refers to the
introduction of foreign DNA into cells. Transfection can be
accomplished by a variety of means known to the art including
calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection, polybrene-mediated transfection, electroporation,
microinjection, liposome fusion, lipofection, protoplast fusion,
retroviral infection, and biolistics. Mammalian cell transfection
techniques are common in the art, and are described in many sources
(See, e.g., Ausubel et al. (eds.), Current Protocols in Molecular
Biology, Vol. 1-4, John Wiley & Sons, Inc., New York
[1994]).
[0085] The term "stable transfection" or "stably transfected"
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant"
refers to a cell which contains stably integrated foreign DNA
within its own genomic DNA.
[0086] The term "transient transfection" or "transiently
transfected" refers to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
transfected cell. The foreign DNA persists in the nucleus of the
transfected cell for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to cells which have taken up foreign DNA but
have failed to integrate this DNA.
[0087] The term "calcium phosphate co-precipitation" refers to a
technique for the introduction of nucleic acids into a eukaryotic
cell, and most typically mammalian cells. The uptake of nucleic
acids by cells is enhanced when the nucleic acid is presented as a
calcium phosphate-nucleic acid co-precipitate. Various
modifications of the original technique of Graham and van der Eb
(Graham and van der Eb, Virol., 52:456 [1973]) are known in which
the conditions for the transfection of a particular cell type has
been optimized. The art is well aware of these various methods.
[0088] The term "transformation" has various meanings, depending on
its usage. In one sense, the term "transformation" is used to
describe the process of introduction of foreign DNA into
prokaryotic cells (i.e., bacterial cells), and most frequently E.
coli strains. Bacterial cell transformation can be accomplished by
a variety of means well known in the art, including the preparation
of "competent" bacteria by the use of calcium chloride, magnesium
chloride or rubidium chloride, and electroporation. When a plasmid
is used as the transformation vector, the plasmid typically
contains a gene conferring drug resistance, such as the genes
encoding ampicillin, tetracycline or kanamycin resistance.
Bacterial transformation techniques are common in the art, and are
described in many sources (e.g., Cohen et al., Proc. Natl. Acad.
Sci. USA 69:2110-2114 [1972]; Hanahan, J. Mol. Biol., 166:557-580
[1983]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory
Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory
Press, NY, [1989]; Ausubel et al. (eds.), Current Protocols in
Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York
[1994]). "Transformation" also describes the physiological process
by which a normal eukaryotic cell acquires the phenotypic qualities
of a malignant cell. Such properties include, but are not limited
to the ability to grow in soft agar, the ability to grow in
nutrient poor conditions, rapid proliferation, and the loss of
contact inhibition. A eukaryotic cell which is "transformed"
displays the properties of malignant cells. In some embodiments,
eukaryotic cells acquire their transformed phenotype in vivo, while
in other embodiments, the cells are artificially transformed in
culture.
[0089] As used herein, the term "selectable marker" refers to the
use of a gene that encodes an enzymatic activity that confers the
ability to grow in medium lacking what would otherwise be an
essential nutrient; in other embodiments, a selectable marker, when
expressed within a cell, confers resistance to an antibiotic or
drug (e.g., the gene encoding the protein which confers resistance
to ampicillin when expressed in a bacterial cells). Furthermore,
some selectable markers are "dominant." Dominant selectable markers
encode an enzymatic activity that is detectable in any suitable
eukaryotic cell line. Examples of dominant selectable markers
include the bacterial aminoglycoside 3' phosphotransferase gene
(i.e., the neo gene) that confers resistance to the drug G-418 in
mammalian cells, as well as the bacterial hygromycin G
phosphotransferase (hyg) gene that confers resistance to the
antibiotic hygromycin, and the bacterial xanthine-guanine
phosphoribosyl transferase gene (i.e., the gpt gene) that confers
the ability to grow in the presence of mycophenolic acid. The use
of non-dominant selectable markers must be in conjunction with a
cell line that lacks the relevant enzyme activity. Examples of
non-dominant selectable markers include the thymidine kinase (tk)
gene (used in conjunction with tk.sup.- cell lines), the CAD gene
(used in conjunction with CAD-deficient cells) and the mammalian
hypoxanthine-guanine phosphoribosyl transferase (hprt) gene (used
in conjunction with hprt.sup.- cell lines). A review of the use of
selectable markers in mammalian cell lines is provided in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory Press, New York (1989), at pp.
16.9-16.15.
[0090] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro.
[0091] As used herein, the term "transformant" refers to organisms
and/or cells capable of harboring an exogenous DNA sequence (e.g.,
via transfection), an expression vector or vehicle, as well as
organisms and/or cells that are suitable for use in expressing a
recombinant gene or protein. It is not intended that the present
invention be limited to any particular type of cell or organism.
Indeed, it is contemplated that any suitable organism and/or cell
will find use in the present invention as a transformant. In some
embodiments, the transformant also transcribes and/or translates
and expresses a gene contained on the exogenous nucleic acid. It is
intended that the exogenous nucleic acid be obtained from any
suitable source. In some embodiments, it is produced synthetically,
while in other embodiments, it is produced by another cell or
organism, and can be recombinant or non-recombinant. In addition,
in some embodiments, the exogenous nucleic acid undergoes
replication, while in other embodiments, it is not. For example,
the bacterium Escherichia coli strain XL-1 Blue is suitable for use
as a transformant for a bacterial expression vector encoding a
ketol-isomerase polypeptide.
[0092] As used herein, the term "prokaryote" refers to organisms
distinguishable from "eukaryotes." It is intended that the term
prokaryote encompass organisms that exhibit the characteristics
indicative of prokaryotes, such as possessing a single, circular
chromosome, the lack of a true nucleus, the lack of membrane-bound
organelles, and other molecular characteristics indicative of
prokaryotes. The term prokaryotes, as used herein, is synonymous
with "bacteria" (i.e., eubacteria, excluding the
archaebacteria).
[0093] As used herein, the term "eukaryote" refers to organisms
distinguishable from "prokaryotes." It is intended that the term
eukaryote encompass all organisms with cells that exhibit the usual
characteristics of eukaryotes such as the presence of a true
nucleus bounded by a nuclear membrane within which reside the
chromosomes, the presence of membrane-bound organelles, and other
characteristics commonly observed in eukaryotic organisms. Thus,
the term eukaryotes includes, but is not limited to, such organisms
as fungi (e.g., molds, sac fungi, club fungi, yeasts),
multicellular photosynthetic plants (e.g., corn, wheat, barley,
soybean, potato, lettuce, rice, tobacco and alfalfa), protozoa
(e.g., Acanthamoeba spp., Trypanosoma spp., Leishmania spp.,
Plasmodium spp., Toxoplasma spp., Giardia spp.), and animals (e.g.,
humans, cattle, sheep, goats, pigs, chickens, turkeys, dogs, cats,
horses, reptiles).
[0094] The term "sample" as used herein is used in its broadest
sense, and can refer to a sample of biological or non-biological
origin. A sample of biological origin refers to any type of
material obtained from animals or plants (e.g., any fluid or
tissue), cultured cells or tissues, cultures of microorganisms
(prokaryotic or eukaryotic), and any fraction or products produced
from a living (or once living) culture. A sample may be a cell
extract (i.e., a crude lysate) or contain intact cells. The
molecule of interest in a sample can be purified or unpurified
(i.e., crude). An "experimental sample" is a sample where the
presence, concentration and/or activity of some molecule of
interest is unknown. A "control sample" is a sample where the
presence, concentration and/or activity of some molecule of
interest is known. For example, control samples containing known
concentrations of some molecule of interest (e.g., glutamate), can
be used to determine the concentration of glutamate in an
experimental sample by using an indicator assay (e.g., the nitro BT
assay) to construct a standard concentration curve. As used herein,
a sample "suspected of containing" a component or
biological/biochemical activity is a sample where the presence of
the component or activity has not been demonstrated or proven.
[0095] As used herein, a "drug" can be any molecule of any
composition, including protein, peptide, nucleic acid, organic
molecule, inorganic molecule, or combinations of molecules,
biological or non-biological, which are capable of producing a
physiological response. As used herein, a "drug" provides at least
one beneficial response in the cure, mitigation, treatment or
prevention of a disease, condition or disorder (e.g., to treat a
microbial infection). A compound is considered a "drug candidate"
if it is not yet known if that compound will provide at least one
beneficial response in the cure, mitigation, treatment or
prevention of a disease, disorder or condition.
[0096] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. The term "in vivo" refers to the natural
environment (e.g., in an animal or in a cell) and to processes or
reactions that occur within a natural environment. The definition
of an in vitro versus in vivo system is particular for the system
under study. For example, as used herein, assays for determining
antifungal activity using the agar diffusion method is an in vitro
assay system. Conversely, the determination of antifungal activity
using a whole mouse candidiasis/candidosis model is an in vivo
assay system.
[0097] As used herein, the term "subject" refers to any animal or
plant being examined, studied or treated. It is not intended that
the present invention be limited to any particular type of subject.
It is contemplated that multiple organisms will find use in the
present invention as subjects. In some embodiments of the present
invention, humans are the preferred subject. Other animals may also
be subjects, including but not limited to cattle, sheep, goats,
pigs, chicken, turkeys, dogs, cats, horses and reptiles). In other
embodiments, plants are the subjects for treatment (e.g., corn,
wheat, barley, soybean, potato, lettuce, rice, tobacco and
alfalfa).
[0098] As used herein, the term "inhibit" refers to the act of
diminishing, suppressing, alleviating, preventing, reducing or
eliminating. The term "suppress" may be used interchangeably with
"inhibit." For example, a compound which inhibits microbial growth
may completely cure (kill all microbial cells, i.e., bacteriocidal
or fungicidal action), prevent microbial growth, arrest or slow
further microbial growth (i.e., bacteriostatic or fungistatic
action). The term "inhibit" can also be used to describe the effect
of a compound on an enzymatic activity. Thus, the term "inhibit" as
it applies to the analysis of enzymatic activity encompasses a
range of effects, from completely eliminating to partially
reducing. The term "inhibit" can be applied to both in vitro as
well as in vivo systems.
[0099] As used herein, the term "bacteriocidal" refers to an agent
that kills bacteria. However, a bacteriocidal agent is not
necessarily able to kill bacterial spores. As used herein, the term
"fungicidal" refers to an agent that kills fungi.
[0100] As used herein, the terms "bacteriostatic" and "fungistatic"
refer to agents which slow or arrest the growth of bacteria and
fungi, respectively, but do not kill the organism.
[0101] As used herein, the terms "local" and "localized" and the
like refer to confinement to a small area or a single tissue (e.g.,
a small area on the skin). The term "localized delivery" is
delivery of an agent (e.g., an antimicrobial compound) to a small
area or single tissue. Localized delivery of an antimicrobial agent
to the skin is termed "topical" delivery. A microbial infection may
be "localized," (i.e., the infection is confined to a relatively
small area or a single tissue, such as the skin), or may be
"systemic" (i.e., the infection has spread to multiple sites,
tissues or organs in a subject, typically via the circulatory or
lymphatic systems). Similarly, delivery of an antimicrobial agent
can be topical (e.g., where delivery is to the surface of the
skin), or systemic (e.g., where delivery is by the circulatory
system via an intravenous or intraarterial injection, or by
gastrointestinal absorption if taken by mouth).
[0102] As used herein, the term "systemic" refers to multiple
sites, tissues or organs in an organism, or to the entire organism.
Use of the word "systemic" generally indicates involvement of the
circulatory and/or lymphatic systems.
[0103] As used herein, the term "systemic delivery" (in contrast to
localized delivery) involves delivery of an agent (e.g., a drug) to
multiple sites, tissues or organs in an organism, or to the entire
organism via the circulatory system following an intravenous
injection, or via gastrointestinal absorption of an orally
administered agent.
[0104] As used herein, the terms "antimicrobial," "antimicrobial
chemotherapeutic," "antimicrobial drug," "antimicrobial compound"
and "antimicrobial activity," are used in reference to any
compound, substance or agent that inhibits the growth of
microorganisms, including eukaryotic microorganisms (e.g., fungi)
and prokaryotes (e.g., bacteria). Thus, antimicrobial agents
comprise both antifungal and antibacterial agents. It is intended
that the term be used in its broadest sense, and includes, but is
not limited to, compounds that exist naturally (e.g., antibiotics)
or compounds that are produced by artificial means (i.e., by in
vitro chemical synthesis). Antimicrobials can be used with or on a
subject in the treatment of microbial disease, infection,
colonization or other pathology. Antimicrobials include compounds
that kill or eliminate microbial growth as well as compounds that
merely suppress, slow or arrest microbial growth. Antimicrobial
activity can take place in vitro, as well as in vivo.
[0105] As used herein, the terms "antifungal," "antifungal agent,"
"antifungal chemotherapeutic," and "antifungal drug" refer to any
compound, substance or agent used in the treatment of fungal
condition, disease, infection or colonization. It includes
fungicidal as well as fungistatic compounds which act on fungi in
vitro, as well as in vivo. Examples of antifungal agents include
amphotericin B, nystatin, fluconazole, itraconazole, naftifine,
ketoconazole, 5-fluorocytosine and griseofulvin. The antifungals of
the present invention are not limited to any particular mechanism
of action. Nor is an understanding of the mechanism of action
necessary to use the antifungals of the present invention.
[0106] Examples of fungal conditions, diseases and infections
includes, but is not limited to: adiaspiromycosis, aspergillosis,
dermatophytoses, blastomycosis, candidemia, cercosporamycosis,
systemic and superficial candidiasis (i.e., candidosis),
chromoblastomycosis, chromomycosis, coccidioidomycosis,
cryptococcosis, cryptomycosis, dermatomycosis,
entomophthoramycosis, favus (tinea favosa), fusariosis,
geotrichosis, histoplasmosis, hyalohyphomycosis, lobomycosis,
maduramycosis (Madura foot), mycetoma, mucormycosis, mycotic
keratitis, mycotic keratosis, onychomycosis, oomycosis, otomycosis,
paracoccidiomycosis, penicillosis, phaeohyphomycosis, phaeomycotic
cyst, piedras (black piedra, white piedra), pityriasis nigra,
pityriasis versicolor (i.e., tinea versicolor), pneumonia,
protothecosis, rhinosporidiosis, ringworm, sporotrichosis, systemic
mycoses, tinea, torulopsosis, trichomycosis axillaris, and
zygomycosis. These examples are intended to be exemplary only, and
it is not intended that the present invention be limited in scope
by these examples.
[0107] As used herein, the terms "antibacterial," "antibacterial
agent," "antibacterial chemotherapeutic," and "antibacterial drug"
refer to any compound, substance or agent used in the treatment of
bacterial condition, disease, infection or colonization. The term
includes antibacterial activity that occurs in vitro, as well as in
vivo. Antibiotics are antibacterial agents derived from natural
sources (e.g., penicillin produced by the mold Penicillum).
Examples of other antibacterial agents include erythromycin,
tetracycline, rifampin, chloramphenicol, isoniazid, ampicillin,
cephalosporin, gentamycin, kanamycin and tobramycin. The
antibacterials of the present invention are not limited to any
particular mechanism of action. Nor is an understanding of the
mechanism of action necessary to use the antibacterials of the
present invention.
[0108] Examples of bacterial conditions, diseases and infections
includes, but is not limited to: anthrax, botulism, conjunctivitis,
diphtheria, food poisoning, gastroenteritis, gonorrhea, meningitis,
pneumonia, salmonellosis, shigellosis, skin and wound infections,
syphilis, tetanus, tuberculosis, typhus and whooping cough. These
examples are intended to be exemplary only, and it is not intended
that the present invention be limited in scope by these
examples.
[0109] As used herein, the term "antimetabolite" refers to any
substance with a close structural resemblance to another essential
substance (i.e., a metabolite) that is required for normal
physiologic function. Typically, antimetabolites exert growth
inhibitory effects by interfering with the utilization of the
metabolite. For example, 5-fluorocytosine is an antimetabolite with
antifungal activity, and sulfanilamide and isoniazid are
antimetabolite antibacterials.
[0110] As used herein, the term "toxicity" generally refers to
deleterious, impairing or injurious effects caused by a compound on
a cell or organism. Toxicity can display a range of severity from
mild to severe. In the extreme, severe toxicity causes the death of
a cell or organism, where mild toxicity may result in symptoms in
an organism that do not significantly impact the fitness or
well-being of the organism.
[0111] As used herein, "selective toxicity" refers to the
phenomenon where a compound may have toxic effects in one organism
or cell type, but does not have those effects on a second organism
or cell type, or has fewer or milder toxic effects on a second
organism or cell type compared to the first organism or cell type.
For example, most antibacterial compounds (e.g., penicillin) are
effective in treating infections in a subject because of the
selective toxicity displayed by the antibacterial compound, where
the antibacterial compound demonstrates severe toxicity towards the
bacteria in a bacterial infection in a host (i.e., bacteriocidal
activity), but that compound does not show toxicity towards the
subject being treated for the infection (e.g., a human). Selective
toxicity may be relative (i.e., not absolute). For example, a
compound that demonstrates potent antibacterial activity may also
have mild toxic effects (i.e., tolerable, manageable or minimal
effects) on the host subject (e.g., a human) being treated for the
infection. If the mild toxicity experienced by the subject does not
significantly impact the fitness or long-term well-being of the
host organism, such toxicity may be acceptable in the treatment of
the infection.
[0112] As used herein, the term "ketol-isomerase activity" refers
to that enzymatic activity catalyzed by the ketol-isomerase enzyme.
Ketol-isomerase activity is that activity that catalyzes the
formation of glucosamine-6-phosphate (GlcN-6-P) and glutamate (Glu)
from fructose-6-phosphate (Fru-6-P) and glutamine (Gln) (See, FIG.
1). The presence of ketol-isomerase activity in a sample is
indicative of the presence of the ketol-isomerase enzyme (i.e.,
"ketol-isomerase" in that sample. However, the absence of
ketol-isomerase activity in a sample does not imply the absence of
ketol-isomerase enzyme, as the presence of an inhibitor may
suppress the ketol-isomerase activity, although the enzyme is
present. As used herein, the addition, identification or detection
of ketol-isomerase activity implies the presence of ketol-isomerase
enzyme.
DESCRIPTION OF THE INVENTION
[0113] The present invention provides novel and improved methods
for the assay of ketol-isomerase activity. In particularly
preferred embodiments, the present invention provides methods that
utilize the redox-sensitive chromophore nitro blue tetrazolium
chloride (nitro BT). The present invention further provides methods
for drug screening to identify compounds that have the ability to
suppress microbial ketol-isomerase activity. It is contemplated
that compounds that inhibit microbial ketol-isomerase activity also
have antimicrobial activity, and can be used to treat microbial
infections (e.g., fungal and/or bacterial infections). The present
invention also provides methods for the identification of
antimicrobial compounds that have selective inhibit microbial
ketol-isomerase compared to a subject's (e.g., mammalian)
ketol-isomerase activity. Although an understanding of the
mechanism by which a compound inhibits ketol-isomerase activity is
not necessary in order to make or use the present invention, the
structure, function and regulation of this enzyme are described
below. Similarly, an understanding of the mechanism by which a
compound imparts antimicrobial activity is not necessary in order
to make or use the present invention. In addition, it is not
intended that the present invention be limited to any particular
mechanisms of antimicrobial action and/or ketol-isomerase activity
inhibition.
[0114] Ketol-Isomerase
[0115] The enzyme ketol-isomerase
(2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase; E.C.
2.6.1.16 or E.C. 5.3.1.19) is required for the biosynthesis of
chitin, as well as other macromolecules. The enzyme is also known
as glutamine:fructose-6-phosphate amidotransferase or
glucosamine-6-phosphate synthase. The enzyme catalyzes the
formation of glucosamine-6-phosphate (GlcN-6-P) and glutamate (Glu)
from fructose-6-phosphate (Fru-6-P) and glutamine (Gln) (See, FIG.
1).
[0116] Ketol-isomerase activity has been identified and
characterized in humans (McKnight et al., J. Biol. Chem.,
267:25208-25212 [1992]; and Daniels et al., J. Clinical Invest.,
97(5):1235-1241 [1996]), other mammals (Winterburn and Phelps,
Biochem. J., 121:701-709 [1971]; and Zalkin Methods Enzymol.,
113:278-281 [1985]), plants, fungi (Endo et al., J. Bacteriol.,
103:588-594 [1970]; Smith et al, J. Bacteriol., 178:2320-2327
[1996]; Watzele and Tanner, J. Biol. Chem., 264:8753-8758 [1989];
Borgia, J. Bacteriol., 174:384-389 [1992]; Milewski et al., J.
Biol. Chem., 274(7):4000-4008 [1999]; and Etchebehere and Da Costa
Maia, Arch. Biochem. Biophys., 272:301-310 [1989]), and bacteria
(Kornfeld, J. Biol. Chem., 242:3135 [1967]; and Badet et al.,
Biochemistry 26:1940-1948 [1987]). Significant biochemical
distinctions can be made between these ketol-isomerase orthologs.
For example, the bacterial enzyme is not inhibited by the
end-product of the Leloir Pathway, UDP-N-acetylglucosamine
(UDP-GlcNAc; See, FIG. 1; Endo et al., J. Bacteriol., 103:588-594
[1970]). However, ketol-isomerase activity from both fungal and
mammalian sources is feedback-inhibited by UDP-GlcNAc (Endo et al.,
J. Bacteriol., 103:588-594 [1970]). Another important distinction
between fungal and mammalian ketol-isomerases can be made, in that
sensitivity of the fungal enzyme to UDP-GlcNAc is dependent on the
phosphorylation state of the ketol-isomerase (i.e., the enzyme is
only feedback-inhibited when it is phosphorylated). In contrast,
phosphorylation does not play a role in mammalian ketol-isomerase
regulation (McKnight et al., J. Biol. Chem., 267:25208-25212
[1992]; and Selitrennikoff et al., Proc. Natl. Acad. Sci. USA
77:5998-6002 [1980]). Also, the fungal enzyme appears to be a
homodimer of a 76 kDa subunit, while the human enzyme appears to be
in the range of 340-360 kDa (a homotetramer of an .about.80 kDa
subunit), and the bacterial enzyme is monomeric (McKnight et al.,
J. Biol. Chem., 267:25208-25212 [1992]). Representative
ketol-isomerases have been cloned and sequenced and the amino acid
alignments of the predicted proteins have shown that while there
are regions of conserved amino acids, there are also regions of
significant sequence divergence between human, bacterial and fungal
ketol-isomerases (Smith et al., J. Bacteriol., 178:2320-2327
[1996]).
[0117] The enzymatic product of ketol-isomerase, GlcN-6-P, is a
precursor of UDP-N-acetyl glucosamine (UDP-GlcNAc), which itself is
the major intermediate of numerous macromolecules including
glycoproteins, proteoglycans, and glycolipids in mammals, chitin
and mannoproteins in fungi, and peptidoglycan and
lipopolysaccharides in bacteria. It is known that mutations that
inhibit ketol-isomerase activity result in the inhibition of fungal
growth (Katz and Rosenberger, Biochim. Biophys. Acta., 208:452-560
[1970]; and Russell and Srb, Molec. Gene. Genet., 129:77-86
[1974]). Furthermore, compounds that inhibit fungal ketol-isomerase
activity also inhibit C. albicans growth (Milewski et al.,
Antimicrob. Agents Chemo., 35:36-43 [1991]; and Milewski et al.,
Drugs Exp. Clin. Res., 14(7):461-465 [1988]).
[0118] Taken together, these results indicate that ketol-isomerase
activity is required for fungal growth, the structure of fungal and
mammalian ketol-isomerases are different, and microbial and
mammalian ketol-isomerases are regulated through different
mechanisms. Thus, selective inhibitors of fungal ketol-isomerase
activity, which have little or no effect on mammalian
ketol-isomerase activity, can be identified. Therefore, such
compounds are a potential target for the development of antifungal
and antibacterial agents. Furthermore, the lack of chitin in
mammalian cells makes their biosynthesis pathways attractive
targets for antifungal compounds. However, the use of compounds
that selectively inhibit microbial ketol-isomerase activity as
antimicrobial agents does not require an understanding of the
mechanism by which the compound functions in order to make and use
the present invention. Nor is it intended that the present
invention be limited to any particular mechanism.
[0119] Ketol-Isomerase Activity Assays
[0120] Methods to determine ketol-isomerase enzymatic activity have
been previously reported. Some of these methods quantitate
GlcN-6-P, one of the products of the ketol-isomerase reaction.
These methods include the Morgan-Elson assay (Zalkin, Methods
Enzymol., 113:278-281 [1985]), a spectrophotometric assay (Badet et
al., Biochemistry 26:1940-1948 [1987]), and high performance liquid
chromatography (HPLC) procedure (Daniels et al., J. Clin. Invest.,
97:1235-1241 [1996]). However, none of these methods are amendable
for high throughput screening, as they require steps that are not
easily automated (e.g., repeated boiling steps), incorporate toxic
reagents (e.g., hydrochloric acid and acetic acid anhydrate),
require chemically unstable reagents (e.g., acetic acid anhydride)
and/or require specialized equipment (HPLC apparatus). For example,
automation of the Morgan-Elson assay is hampered by multiple
boiling steps and the requirement for 10 N hydrochloric acid,
measurement of UV absorption is needed in the spectrophotometric
assay, and HPLC is an equipment-intensive and expensive
methodology.
[0121] An alternative method for the quantitation of
ketol-isomerase activity in crude cell lysate has also been
described which measures the concentration of glutamate, a
ketol-isomerase reaction by-product, and uses
p-iodonitrotetrazolium violet (Selitrennikoff and Ostroff, Emerging
Therapeutic Targets 3:53-72 [1999]). This iodonitrotetrazolium
violet assay uses a two step enzymatic method requiring glutamate
dehydrogenase (E.C. 1.4.1.3) and diaphorase (E.C. 1.8.1.4). In this
assay, glutamate dehydrogenase first converts glutamate to
.alpha.-ketoglutarate, utilizing the oxidized form of nicotinamide
adenine dinucleotide (.beta.-NAD) as a cofactor. In the second step
of the assay, the enzyme diaphorase reduces p-iodonitrotetrazolium
violet (INV), using .beta.-NADH (the reduced form of .beta.-NAD
produced from the prior enzymatic step). The compound
p-iodonitrotetrazolium violet is a chromophore that reflects red
light following its reduction by .mu.-NADH. The amount of light
produced by the chromophore (measured spectrophotometrically)
following exposure to .beta.-NADH is proportional to the amount of
glutamate present following the ketol-isomerase reaction.
[0122] In this iodonitrotetrazolium violet assay, two exogenous
enzymes (glutamate dehydrogenase and diaphorase) are required.
Therefore, not only ketol-isomerase inhibitors, but also inhibitors
of these exogenous enzymes are detected by this assay, thus, "false
positives" are detected. Furthermore, since two enzymatic reactions
are required in this method, the assay is subject to an added
component of variability. Therefore, establishment of a
ketol-isomerase assay that is amenable to high-throughput
automation (e.g., using microtiter plates), yields minimal false
positive results and shows minimal variability is needed in the art
to provide a tool for screening antimicrobial agents.
[0123] The Nitro BT Ketol-Isomerase Assay of the Invention
[0124] The present invention provides simplified and improved
methods for the assay (i.e., quantitation) of ketol-isomerase
activity by indirectly determining glutamate concentration. As
shown in FIG. 2, GlcN-6-P and glutamate are produced as products of
the ketol-isomerase reaction. The ketol-isomerase reaction is
followed by a heat inactivation step, resulting in the irreversible
denaturation of the enzyme and permanent cessation of further
ketol-isomerase activity. Following the heat inactivation step, a
secondary enzymatic reaction is employed with glutamate
dehydrogenase to produce .alpha.-ketoglutarate and .beta.-NADH from
the glutamate (produced in the ketol-isomerase reaction) and a
molar excess of .beta.-NAD. The .beta.-NADH produced in this
secondary reaction is then quantitated using a redox-sensitive
chromogenic substrate.
[0125] The reduction-sensitive chromophore used in this method is
nitro blue tetrazolium chloride (nitro BT). Nitro BT is reduced in
the presence of .beta.-NADH and phenazine methosulfate (PMS) to
yield a compound that is measurable at a 585 nm wavelength, and is
readily quantitated using standard colorimetric spectrophotometry
(Van Noorden and Butcher, Anal. Biochem., 176:170-174 [1989]). A
schematic of this reaction is shown in FIG. 2. The use of nitro BT
to indirectly measure ketol-isomerase enzymatic activity by
quantitating the accumulation of the secondary reaction product
(.beta.-NADH) is novel. The nitro BT assay is applicable to the
assay of ketol-isomerase activity from any organism, and as shown
below, is equally or more sensitive than known ketol-isomerase
activity assays. Furthermore, in the nitro BT assay, only one
supplemental enzymatic reaction is used in the assay (i.e., the
glutamate dehydrogenase reaction). Thus, the number of false
positives and interassay variability are reduced.
[0126] The nitro BT assay method was first tested to determine
whether the assay provided a linear response using known glutamate
concentration standards. The detailed conditions of this experiment
are described in Example 1. Briefly, aliquots of glutamate standard
solutions ranging in concentration from 0 to 600 .mu.M were placed
in the wells of a 96 well microtiter plate and combined with the
reaction buffer containing glutamate dehydrogenase enzyme and molar
excesses of NAD, PMS, and nitro BT. The mixture was incubated for
15 min at room temperature, followed by spectrophotometric analysis
at 585 nm.
[0127] The results of this experiment are shown in FIG. 3, where
the plot depicts the OD.sub.585 nm as a function of glutamate
standard concentration. Each data point indicates the mean
OD.sub.585 nm of triplicate samples.+-.the standard deviation
(S.D.). As indicated in FIG. 3, glutamate concentrations within
this range yielded a linear response with respect to the
calorimetric detection of the reduced form of the nitro BT. This
indicates a direct correlation between glutamate concentration and
optical density, thus verifying that OD is an appropriate measure
of glutamate concentration following the glutamate dehydrogenase
and nitro BT reactions.
[0128] Side-By-Side Comparison of Nitro BT and Morgan-Elson
Assays
[0129] Following verification of the nitro BT assay to quantitate
glutamate, suitability of the assay to quantitate ketol-isomerase
activity was addressed in a side-by-side comparison with the
Morgan-Elson assay to quantitate glutamate and GlcN-6-P
concentrations, respectively, following a ketol-isomerase reaction.
The source of the ketol-isomerase activity was a crude cell extract
prepared from Aspergillus fumigatus hyphae. The specific
experimental conditions used in this comparison are provided in
Examples 1, 4 and 5.
[0130] Briefly, to prepare the cell extract, A. fumigatus conidia
were used to inoculate a liquid culture and incubated. Hyphae were
harvested by vacuum filtration, washed, snap-frozen, and stored at
-80.degree. C. until use. The frozen A. fumigatus hyphae were
thawed and disrupted by bead-beating using 0.5 mm zirconium beads
in physiological sucrose buffer. The resulting lysates were
centrifuged to remove cell debris, and supernatants were stored at
-80.degree. C. until ready for use in the ketol-isomerase
assay.
[0131] Each ketol-isomerase reaction mixture contained 0.3 mg
protein/mL of the cell lysate and molar excesses of
fructose-6-phosphate (Fru-6-P) and glutamine. The reactions were
incubated at 37.degree. C. for 20, 40 or 60 minutes, and then were
terminated by boiling for 5 minutes, or alternatively, by heating
to 70.degree. C. for 15 minutes. The reaction mixtures were then
centrifuged, and the clarified supernatants were used in the
Morgan-Elson and nitro BT assays.
[0132] As discussed above, the ketol-isomerase reaction catalyzes
the formation of glucosamine-6-phosphate (GlcN-6-P) and glutamate
(Glu) from fructose-6-phosphate (Fru-6-P) and glutamine (Gln) (as
shown in FIG. 2). The two end products of this in vitro reaction,
namely GlcN-6-P and glutamate, were quantitated using the
Morgan-Elson and nitro BT assays, respectively.
[0133] A modified Morgan-Elson assay was used to determine GluN-6-P
concentration following the ketol-isomerase reaction (as described
in Example 4). Briefly, saturated NaHCO.sub.3 solution and acetic
acid were combined with the ketol-isomerase reaction supernatant.
The mixture was incubated at room temperature for 3 minutes, boiled
for 3 minutes, followed by the addition sodium borate solution,
boiled again for 8 minutes, then finally cooled to room
temperature. Ehrlich's reagent (See, Example 4) was then added to
each reaction, followed by incubation at 37.degree. C. for 20
minutes and colorimetric analysis (OD.sub.585 nm). In the
Morgan-Elson assay, color development is directly proportional to
GluN-6-P concentration. Samples of GluN-6-P of known concentration
were analyzed in parallel to provide standardization values in
order to determine absolute GluN-6-P concentration in the
experimental samples.
[0134] Determination of glutamate concentrations was also done
using the nitro BT assay of the present invention, as described
above, and in Examples 1 and 5. In this experiment, samples of
glutamate of known concentration were analyzed in parallel, to
provide standardization values, in order to determine absolute
glutamate concentration in the experimental samples.
[0135] Finally, the GlcN-6-P and glutamate concentrations
determined by the Morgan-Elson and nitro BT assays, respectively,
were plotted on the same graph over the time course of the
ketol-isomerase reaction. The results of this side-by-side
comparison are shown in FIG. 4. If the Morgan-Elson and nitro BT
assays are equally sensitive, it was predicted that the
concentration curves for GlcN-6-P and glutamate during the
ketol-isomerase reaction would be identical, since the GlcN-6-P and
glutamate are produced by the ketol-isomerase reaction in a 1:1
stoichiometric ratio. As indicated in FIG. 4, these two different
methods yielded nearly identical concentrations of these two
reaction products. In a control ketol-isomerase reaction that
omitted glutamine, neither glutamate nor GlcN-6-P were detected
(data not shown).
[0136] From these results, it is clear that the nitro BT assay of
the present invention is able to quantitate ketol-isomerase
activity at least as effectively as the Morgan-Elson assay. In
addition, the present invention provides advantages over current
methods for the identification of inhibitors of ketol-isomerase, as
the methods of the present invention have fewer and simplified
steps compared to those known in the art (e.g., the Morgan-Elson
assay).
[0137] Detection of Inhibitors of Ketol-Isomerase Using the Nitro
BT Assay
[0138] It is contemplated that compounds that inhibit microbial
ketol-isomerase activity will find use as antimicrobials suitable
for use in treatment of microbial infections and disease. It is
further contemplated that the nitro BT assay of the present
invention will find widespread use in the identification of
compounds that have the ability to inhibit the activity of fungal
or bacterial ketol-isomerase. Furthermore, the nitro BT assay of
the present invention is readily adapted for use in high throughput
drug screening. In addition, the present invention provides methods
for the identification of undeveloped and/or unknown antimicrobial
agents. Also, use of the antimicrobial drugs identified using the
methods of the present invention are less likely to be subject to
microbial resistance.
[0139] In order to demonstrate the ability of the nitro BT assay to
detect compounds having the ability to suppress ketol-isomerase
activity, a known inhibitor of ketol-isomerase was included in an
A. fumigatus ketol-isomerase reaction assay, as described in
Example 6. In this experiment, UDP-N-acetylglucosamine (UDP-GlcNAc)
was added to the ketol-isomerase reaction at a concentration range
from 0 to 10 mM. UDP-GlcNAc is known to cause feedback inhibition
of Neurospora crassa ketol-isomerase (Endo et al., J. Bacteriol.,
103:588-594 [1970]).
[0140] Briefly, in this experiment, a series of ketol-isomerase
reactions were analyzed in parallel, wherein each reaction mixture
contained Fru-6-P, glutamine, and crude A. fumigatus cell extract
(as described in Example 3). In addition, reactions also contained
various concentrations of UDP-GlcNAc, ranging from 0 to 10 mM.
Reactions were incubated at 37.degree. C. for 60 minutes, after
which time the ketol-isomerase reactions were terminated by heating
at 70.degree. C. for 15 min. However, the ketol-isomerase reaction
can also be terminated by boiling for 5 minutes. In addition, the
ketol-isomerase reaction can be clarified following the
inactivation step by centrifugation or filtration.
[0141] Following arrest of the ketol-isomerase reaction, the
necessary reagents for the nitro BT reaction were added to each
reaction mixture, including glutamate dehydrogenase enzyme and
molar excesses of G-NAD, PMS, and nitro BT. After incubation at
37.degree. C. for 90 minutes, the color development was quantitated
by measurement of OD.sub.585 nm .
[0142] The results of this assay are shown in a bar-graph format in
FIG. 5. Each data point represents the mean of three samples; the
standard deviation of each data point is indicated by a vertical
line. As indicated in FIG. 5, the ketol-isomerase activity
contained in the A. fumigatus crude extract was inhibited by
UDP-GlcNAc in a dose-dependent manner. The IC.sub.50 (concentration
of a compound which inhibits enzymatic activity to 50% relative to
an untreated control) was 0.48 mM. This inhibitory effect is
similar to that previously observed with UDP-GlcNAc in the
inhibition of ketol-isomerase activity from C. albicans strain ATCC
No. 10261 crude cell extracts (IC.sub.50=0.69 mM; Milewski et al.,
J. Biol. Chem., 274:4000-4008 [1999]). These results indicate that
ketol-isomerase inhibitors can be successfully detected using the
nitro BT assay of the present invention.
[0143] Although the present invention was reduced to practice using
crude cell lysates from the fungus A. fumigatus as a source of
ketol-isomerase activity, it is not intended that the invention be
limited to this particular fungal species. Indeed, the present
invention finds use with crude cell lysates of numerous fungal
species, especially pathogenic fungal species. In addition,
non-pathogenic fungal species also find use with the present
invention as a source of ketol-isomerase activity (e.g.,
Saccharomyces cerevisiae and Schizosaccharomyces pombe). Thus, the
present invention encompasses use with various fungi, including but
not limited to:
[0144] Absidia spp. (e.g., A. corymbifera),
[0145] Acremonium spp. (e.g., A. falciforme, A. kiliense, and A.
recifei),
[0146] Aspergillus spp. (e.g., A. flavus, A. fumigatus, A. niger,
A. versicolor, A. nidulans, A. sydowii, A. terreus and A.
flavipes),
[0147] Blastomyces spp. (e.g., B. dermatitidis),
[0148] Candida spp. (e.g., C. albicans, C. tropicalis, C. kefyr, C.
guilliermondii, C. krusei and C. parapsilosis, C. glabrata, C.
catenulata, C. ciferrii, C. haemulonii, C. lipolytica, C.
lusitaniae, C. novegensis, C. pulcherrima, C. rugosa, C. utilis, C.
viswanathii and C. zeylanoides),
[0149] Cladosporium werneckii,
[0150] Claviceps purpura,
[0151] Coccidioides immitis,
[0152] Cryptococcus spp. (e.g., C. neoformans, C. albidus, C.
laurentii, C. luteolus, C. terreus and C. uniguttulatus),
[0153] Epidermophyton spp. (e.g., E. floccosum),
[0154] Fusarium spp.,
[0155] Histoplasma spp. (e.g., H. capsulatum),
[0156] Microsporum spp.,
[0157] Mucor spp.,
[0158] Paracoccidioides brasiliensis,
[0159] Philophora verrucosa,
[0160] Peidraia hortai,
[0161] Pityrosporum orbiculare,
[0162] Pneumocystis spp. (e.g., P. carinii),
[0163] Rhizomucor spp. (e.g., R. pusillus),
[0164] Rhizopus spp. (e.g., R. microsporus, R. arrhizus and R.
oryzae),
[0165] Saccharomyces spp. (e.g., S. cerevisiae),
[0166] Schizosaccharomyces spp. (e.g. S. pombe),
[0167] Sporothrix schenckii,
[0168] Trichophyton spp., and
[0169] Trichosporon beigelii.
[0170] Of the species in this list, Aspergillus spp., Candida spp.,
Cryptococcus spp., Histoplasma spp., Pneumocystis spp., Rhizopus
spp., Saccharomyces spp. (e.g., S. cerevisiae) and
Schizosaccharomyces spp. (e.g., pombe) find particular use with the
invention. However, the above list is intended to be exemplary
only, and is not intended to limit the scope of the invention, nor
limit the sources of ketol-isomerase used with the present
invention.
[0171] Furthermore, bacterial sources of ketol-isomerase activity
also find use with the present invention. In one embodiment,
bacterial extracts containing ketol-isomerase activity find use in
identifying compounds that have the ability to suppress bacterial
ketol-isomerase activity, and thus, can identify compounds with
antibacterial activity. The preparation of suitable bacterial
extracts are common in the art, and protocols to prepare such
extracts can be found in numerous sources (See, e.g., Ausubel et
al. (eds.), Current Protocols in Molecular Biology, "Analysis of
Protein Interactions," p. 20.2.1-20.2.10, John Wiley & Sons,
Inc., New York [1994]). In one such protocol, Escherichia coli can
be grown overnight in 1.0 L LB liquid culture to generate starting
material to prepare bacterial cell lysate. The cells are harvested
by centrifugation, and the resulting pellet is washed with a buffer
such as PBS or other physiological salt buffer (e.g., a buffer
containing 50 mM potassium phosphate, 150 mM potassium chloride and
1 mM magnesium chloride, pH 7.5). After washing, the cells are
pelleted again, and resuspended in 10 mL of the potassium phosphate
buffer described above, and subjected to sonication using a
microprobe for a series of 5.times.1 minute intervals. After the
sonication, the cells are centrifuged to remove the cell debris,
the supernatant is recovered, and the protein concentration of the
resulting cell lysate is determined. The lysate can be optionally
stored with glycerol, a detergent such as Triton X-100, and/or
protease inhibitors. In preferred embodiments, lysates from
pathogenic bacterial species are used (e.g., Staphylococcus aureus
and Pseudomonas aeruginosa). It is not intended that the present
invention be limited to the use of E. coli as a source of
ketol-isomerase activity, as any other bacterial species that
produces ketol-isomerase finds use with the present invention. For
example, the present invention finds use with various bacterial
species, including but not limited to:
[0172] Bacillus spp. (e.g., B. anthracis),
[0173] Bordetella pertussis,
[0174] Brucella spp.,
[0175] Campylobacter spp.,
[0176] Chlamydia spp.,
[0177] Clostridium spp.,
[0178] Enterococcus spp. (e.g., E. faecalis, E. faecium, E.
mundtii, E. hirae, E. casseliflavus and E. gallinarum),
[0179] Escherichia spp. (E. coli),
[0180] Haemophilus spp. (e.g., H. influenzae),
[0181] Helicobacter spp. (e.g., H. pylori),
[0182] Klebsiella spp.,
[0183] Mycobacterium spp.,
[0184] Neisseria spp.,
[0185] Pseudomonas spp. (e.g., P. aeruginosa, P. fluorescens, P.
stutzeri, P. oryzihabitans, P. luteola, P. alcaligene and P.
mendocina),
[0186] Rickettsia spp.,
[0187] Salmonella spp.,
[0188] Serratia marcescens,
[0189] Shigella spp.,
[0190] Staphylococcus spp. (e.g., S. aureus, S. epidermitis, S.
saprophyticus, S. haemolyticus, S. capitis, S. caprae, S.
lugdunensis, S. saccharolyticus, S. simulans, S. warneri, S.
hominis, S. schleiferi and S. intermedius),
[0191] Streptobacillus spp.,
[0192] Streptococcus spp. (e.g., S. pyogenes, S. agalactiae, S.
pneumoniae, S. sanguis, S. mitis, S. oralis, S. gordonii, S.
intermidius, S. anginosus, S. salivarius and S. bovis),
[0193] Treponema spp.,
[0194] Vibrio spp., and
[0195] Yersinia spp.
[0196] Of the species in this list, Escherichia coli,
Staphylococcus spp. and Pseudomonas spp. find particular use with
the invention. However, the above list is intended to be exemplary
only, and is not intended to limit the scope of the invention, nor
limit the sources of ketol-isomerase used with the present
invention.
[0197] Furthermore, purified enzymatic preparations obtained using
the methods of the present invention find use as a source of
ketol-isomerase activity suitable for use in the ketol-isomerase
reaction. Such preparations can be derived from any species (e.g.,
bacteria, fungi or animals), and the method of purification is not
limited to any particular method. Indeed, numerous techniques are
known in the art for polypeptide purification (e.g., ammonium
sulfate precipitation, gel chromatography, ion exchange
chromatography, affinity chromatography, etc.). These techniques
may be used independently or in an appropriate combination to
purify a ketol-isomerase polypeptide from a cell lysate starting
culture.
[0198] In alternative embodiments, protocols employing molecular
genetics techniques are used to overexpress a recombinant
ketol-isomerase protein (derived from any species) in E. coli,
followed by harvesting of the E. coli and preparation of a crude
cell lysate for use in the ketol-isomerase reaction. Alternatively
still, in some embodiments, the crude cell lysate collected from
the E. coli overexpressing the recombinant ketol-isomerase protein
is used as starting material for purification or enrichment of the
protein, and that purified protein is suitable for use in the
ketol-isomerase assay. In still other embodiments, fusion protein
constructs are used to generate a recombinant chimeric protein,
where the chimeric protein has a fused "tag" that facilitates
purification of the protein. Examples of fusion protein methods
include fusions with glutathione-S-transferase (GST), maltose
binding protein (MBP), polyhistidine tags (i.e., 6.times.His), and
thioredoxin tags. Numerous protocols for the purification of
proteins, the use of bacterial overexpression systems, and
construction of chimeric fusion proteins are widely known to those
skilled in the art (See, e.g., Ausubel et al. [eds.], Current
Protocols in Molecular Biology, Sections 10.9-10.11, and 16.1-16.8,
John Wiley & Sons, Inc., New York [1994]). Furthermore, methods
for the cloning, genetic manipulation and purification of
ketol-isomerase enzyme from bacterial, fungal and mammalian species
are also known (Badet et al., Biochemistry 26:1940-1948 [1987];
McKnight et al., J. Biol. Chem., 267:25208-25212 [1992]; Milewski
et al., J. Biol. Chem., 274:4000-4008 [1999]; Smith et al., J.
Bacteriol., 178:2320-2327 [1996]; Watzele and Tanner, J. Biol.
Chem., 264:8753-8758 [1989]; Winterburn and Phelps, Biochem. J.,
121:701-709 [1971]; and Zalkin Methods Enzymol., 113:278-281
[1985]).
[0199] Further still, it is not intended that the present invention
be limited to the use of a full length ketol-isomerase protein in
these enzymatic assay methods, as either full length or truncated
ketol-isomerase proteins find use in these in vitro assays.
However, when truncated enzymes are used in these assays, these
enzymes must exhibit ketol-isomerase activity.
[0200] Thus, the drug screening methods provided by the present
invention find use with crude cell extracts (i.e., lysates), as
well as or purified protein fractions, and the source of the
ketol-isomerase activity is not limited to any particular source.
Therefor, ketol-isomerases of any species find use with the methods
of the present invention, even though their respective
ketol-isomerase proteins or genes have not been purified and/or
isolated. It is not intended that the present invention be limited
to any particular species as a source of ketol-isomerase activity,
any particular method for preparing a cell lysate, nor any
particular method for enzyme purification or enrichment, as those
in the art will recognize numerous equivalent methods that find use
with the present invention.
[0201] The present invention provides methods for the
identification of compounds having the ability to inhibit microbial
ketol-isomerase activity. Compounds having the ability to inhibit
ketol-isomerase activity are candidates for further development as
antimicrobial agents. In the methods provided by the present
invention, a series of ketol-isomerase reactions are analyzed,
where each ketol-isomerase reaction mixture contains Fru-6-P,
glutamine, and a source of microbial ketol-isomerase (as described
in Example 3). In addition, reactions also containing the test
compound are analyzed in parallel. Reactions are incubated at
37.degree. C. for 60 minutes, after which time the ketol-isomerase
reactions are terminated, The conditions of 37.degree. C. and 60
minutes are intended to be exemplary only, and are not intended to
limit the methods of the present invention, as other conditions
also find use with the invention. All reactions, including those
reactions containing the test compound, are conducted under
conditions where ketol-isomerase is capable of producing GlcN-6-P
and glutamate (e.g., 37.degree. C. for 60 minutes). In the event
that the test compound is a strong inhibitor of ketol-isomerase
activity, little or no GlcN-6-P and glutamate are produced, despite
the fact that the reaction was conducted under conditions that
would otherwise result in the formation of GlcN-6-P and
glutamate.
[0202] Identification of Selectively Toxic Antimicrobial
Compounds
[0203] In a most preferred embodiment, the present invention
provides methods for identifying compounds that selectively inhibit
microbial ketol-isomerase activity relative to mammalian
ketol-isomerase activity. The compounds identified in these
methods, thereby, are antimicrobial compounds that have no toxic
effects on a host when administered to treat (i.e., eradicate,
ameliorate or prevent) a microbial infection. In another
embodiment, the antimicrobial compounds identified in the methods
of the present invention display selective toxicity, in which the
compound having potent antimicrobial activity has fewer or mild
toxic effects (i.e., tolerable, manageable or minimal effects) on
the host subject (e.g., a human) being treated for the infection.
In this embodiment, the mild toxicity experienced by the subject
being treated does not significantly impact the fitness or
long-term well-being of the subject, and such toxicity may be
acceptable in the treatment of the infection. In a most preferred
embodiment, the subject is a human, although the antimicrobial
compounds identified in these methods can be used to treat any
subject, including but not limited to other mammalian and
non-mammalian animals (e.g., cattle, sheep, goats, pigs, chickens,
turkeys, dogs, cats, horses and reptiles) and plants (e.g., corn,
wheat, barley, soybean, potato, lettuce, rice, tobacco and
alfalfa).
[0204] Thus, the present invention provides methods for the
identification of compounds having selective toxicity towards
microbial species, but are not toxic or are less toxic (i.e.,
minimally toxic) towards the subject species (e.g., animals or
plants). As described above, the nitro BT assay of the present
invention finds use in the identification of compounds that inhibit
microbial (e.g., bacterial or fungal) ketol-isomerase activity. In
some embodiments, following the identification of such a compound,
the compound is then tested in a ketol-isomerase activity assay
using a second source of ketol-isomerase, to determine whether the
compound has inhibitory activity towards the second
ketol-isomerase, where the second source of ketol-isomerase is from
a species that is a potential subject (e.g., a higher plant or
animal). In a preferred embodiment, a compound that has inhibitory
activity towards a microbial ketol-isomerase will have less or no
inhibitory activity towards a subject's ketol-isomerase (i.e., the
compound preferentially inhibits the microbial ketol-isomerase
activity compared to the subject's ketol-isomerase activity). Thus,
the present invention provides means to identify compounds that
inhibit microbial ketol-isomerase(s), but do not inhibit or inhibit
to a lesser extent a subject's ketol-isomerase(s), for use as
antimicrobials, and have minimal or no toxicity when administered
to a subject.
[0205] In preferred embodiments, a mammalian ketol-isomerase is
used; however, it is not intended that the source of the mammalian
ketol-isomerase be limited. In particularly preferred embodiments,
the mammalian ketol-isomerase is a human ketol-isomerase.
Furthermore, in most preferred embodiments, the source of human
ketol-isomerase is a recombinant human ketol-isomerase expressed in
host bacteria, and a crude cell extract (i.e., cell lysate) of that
bacteria is used in the ketol isomerase assay. In other
embodiments, the recombinant human ketol-isomerase in the bacterial
cell extract is purified and/or enriched prior to use in the
ketol-isomerase assay.
[0206] The cloning, purification and use of crude or purified
mammalian ketol-isomerase preparations is known in the art
(McKnight et al., J. Biol. Chem., 267:25208-25212 [1992]; Daniels
et al., J. Clinical Invest., 97(5):1235-1241 [1996]; Winterburn and
Phelps, Biochem. J., 121:701-709 [1971]; and Zalkin Methods
Enzymol., 113:278-281 [1985]). In a particularly preferred
embodiment of the present invention, a cloned human ketol-isomerase
cDNA overexpressed in E. coli, which are used to produce a crude
bacterial lysate containing human ketol-isomerase activity (as
described in McKnight et al., J. Biol. Chem., 267:25208-25212
[1992]) are used. In this protocol, the open reading frame encoding
the human ketol-isomerase is subcloned into a vector (pPROK-1;
Clontech) permitting inducible expression in bacterial cells (E.
coli XL-1 Blue; Stratagene) via an operably linked tac promoter.
The transformed bacteria are grown in liquid culture, and
expression of the human ketol-isomerase is induced by the addition
of isopropylthio-.beta.-galactopyranoside (IPTG) for two hours.
Cell pellets are harvested by centrifugation, then resuspended in
lysis buffer (100 mM NaH.sub.2PO.sub.4 [pH 7.5], 50 mM KCl, 10 mM
EDTA, 12 mM glucose-6-phosphate and 100 mM phenylmethylsulfonyl
fluoride [PMSF]). Cells are lysed by sonication, and the crude
cytosol is recovered by centrifugation. This crude lysate is used
directly in the ketol-isomerase reaction (with and without the
compound being tested), followed by the nitro BT assay to determine
whether the compound has the ability to inhibit the human
ketol-isomerase activity.
[0207] In an alternative embodiment, human ketol-isomerase activity
is obtained by harvesting a crude lysate from cultured human cells
(as described in Daniels et al., J. Clinical Invest.,
97(5):1235-1241 [1996]). In this protocol, human cells (e.g.,
primary myotubes from human muscle biopsy) are cultured, followed
by harvesting, lysing by sonication, then centrifuging to obtain
the crude human cell lysate containing ketol-isomerase
activity.
[0208] In still another alternative embodiment of the present
invention, a purified preparation of rat ketol-isomerase is used in
the ketol-isomerase reaction (as described in Winterburn and
Phelps, Biochem. J., 121:701-709 [1971]; and Zalkin Methods
Enzymol., 113:278-281 [1985]). In this protocol, rat livers are
obtained from sacrificed animals, and placed in a homogenization
buffer (50 mM Tris, 5 mM EDTA, 5 mM glutathione, 5 mM
glucose-6-phosphate, and 100 mM KCl, pH 7.8). The tissue is
homogenized in a mechanical homogenizer, then followed by low-speed
and high-speed centrifugation steps to remove cell debris. The
supernatant is recovered, and subjected to two DEAE-cellulose
column chromatography steps, and an hydroxyapatite fractionation.
Ammonium sulphate fractionation may also be incorporated into the
purification protocol. These methods of protein purification are
described in many sources, and are known to those familiar with the
art.
[0209] Assays for Antimicrobial Activity
[0210] In some embodiments, compounds identified as being capable
of inhibiting microbial ketol-isomerase activity also have
antimicrobial activity, and are suitable for use in the treatment
of microbial infections. In one embodiment of the present
invention, compounds able to inhibit microbial ketol-isomerase are
tested for their ability to inhibit microbial growth. Inhibition of
microbial growth can include the complete killing and elimination
of microbial cells, the slowing of the rate of growth of microbial
cells, or the arrest of microbial metabolism. Numerous methods are
known in the art for the assessment of antimicrobial activity (See
e.g., Turnidge and Jorgensen and Kennedy, Ch. 115, "Antimicrobial
Susceptibility Testing: General Consideration," Jorgensen et al.,
Ch. 118, "Antibacterial Susceptibility Tests: Dilution and Disk
Diffusion Methods," Ferraro and Jorgensen, Ch. 123, "Susceptibility
Testing Instrumentation and Computerized Expert Systems for Data
Analysis and Interpretation," Inderlied and Salfinger, Ch. 124,
"Antimycobacterial Agents and Susceptibility Tests," and
Espinel-Ingroff et al., Ch. 126, "Antifungal Agents and
Susceptibility Tests," all in Murray et al., (eds), Manual of
Clinical Microbiology, 7th ed., ASM Press, Washington, D.C. [1999];
and National Committee for Clinical Laboratory Standards [NCCLS],
"Reference Method for Broth Dilution Susceptibility Testing of
Yeasts, Publication M27-A [1997]). Thus, it is not intended that
the present invention be limited to any particular method for
screening for antimicrobial activity, as numerous antimicrobial
activity assays find use with the present invention, as known to
those familiar with the art.
[0211] In some embodiments of the present invention, compounds
having the ability to inhibit bacterial ketol-isomerase activity
are tested for antibacterial activity using in vitro assays.
Methods to test the antibacterial activity of a compound are common
in the art, and include dilution susceptibility assays (broth and
agar methods), and agar diffusion assays (e.g., disk diffusion),
(See e.g., National Committee for Clinical Laboratory Standards
[NCCLS], "Methods for Dilution Antimicrobial Susceptibility Tests
for Bacteria That Grow Aerobically," Publication M7-A4 [1997]; and
Jorgensen et al., Ch. 118, p. 1526-1543, "Antibacterial
Susceptibility Tests: Dilution and Disk Diffusion Methods," in
Murray et al., (eds), Manual of Clinical Microbiology, 7th ed., ASM
Press, Washington, D.C. [1999]). In some embodiments, these methods
are adapted to provide for high throughput screening of large
numbers of compounds. A variety of bacterial species find use in
the in vitro antimicrobial testing methods. In some embodiments,
known reference strains that are genetically stable are used in
these methods, including but not limited to E. coli (e.g., ATCC
Strain No. 25922), S. aureus (e.g., ATCC Strain) and P. aeruginosa
(e.g., ATCC Strain No. 27853).
[0212] In other embodiments of the present invention, compounds
having the ability to inhibit fungal ketol-isomerase activity are
tested for antifungal activity using various in vitro assays.
Methods to test the antifungal activity of a compound are common in
the art, and include broth macrodilution and broth microdilution
assays, agar diffusion assays (e.g., disk diffusion), and
agar-dilution assays (See, e.g., National Committee for Clinical
Laboratory Standards [NCCLS], "Reference Method for Broth Dilution
Susceptibility Testing of Yeasts," Publication M27-A [1997]). In
some embodiments, these methods are adapted to provide for high
throughput screening of large numbers of compounds. In some
embodiments, known reference strains of fungal species, including
but not limited to Candida spp. (e.g., C. parapsilosis ATCC Strain
No. 22019 and C. krusei ATCC Strain No. 6258) and Aspergillus spp.
are used in in vitro testing.
[0213] In addition, it is not intended that the invention be
limited to the use of any one particular microbial growth
inhibition assay and/or antimicrobial testing method. Indeed,
numerous methods find use with the present invention. Similarly, it
is not intended that the present invention be limited to assays
utilizing Candida spp., Aspergillus spp., E. coli, S. aureus or P.
aeruginosa, as these species are only exemplary, and other
microbial species also find use with the present invention.
[0214] In addition, in some preferred embodiments of the present
invention, compounds which demonstrate an ability to inhibit
microbial ketol-isomerase activity in vitro and have antimicrobial
properties in vitro are tested in an in vivo animal models of
disease. For example, in some preferred embodiments, the mouse
model of candidosis/candidemia is used. This fungal infection model
is known in the art (Hanson et al., Antimicrob. Agents Chemother.,
35:1334-1337 [1991]).
[0215] Briefly, two groups of five-week-old female CD-1 mice
(Charles River Laboratories) are infected intravenously with a
wild-type C. albicans strain. Concurrently, the mice in one of the
groups are also injected with a test compound suspected of having
antimicrobial activity (as determined in vitro). Mouse mortality is
scored over 14 days post-infection. Alternatively, mice are
injected with the test compound after an interval of time following
the original inoculation with C. albicans. This alternative
protocol allows the determination of whether a compound is capable
of eliminating infection (i.e., as opposed to preventing the
infection).
[0216] It is not intended that the invention be limited to the use
of one particular in vivo infection model. It is contemplated that
other in vivo infection assays will be used with the present
invention. Indeed, it is contemplated that any method suitable to
determine the in vivo effectiveness of an antimicrobial compound or
combination of compounds will find use in the present invention.
For example, a mouse model of aspergillosis using A. fumigatus
finds use with the present invention. This method is known in the
art, and uses a method nearly identical to that described above for
the mouse candidemia model, with the exception that A. fumigatus is
used as the mouse inoculum. It is also not intended that the
present invention be limited to use in identifying compounds
effective in the treatment of C. albicans or A. fumigatus
infections, as the present invention provides methods suitable for
the identification of antimicrobial compounds effective in the
treatment of numerous microbial infections.
[0217] In some preferred embodiments of the present invention,
candidate antimicrobial compounds advance through sequential
screening steps in order to identify promising compounds for
further clinical development. In particularly preferred
embodiments, the identification of candidate therapeutic compounds
is conducted in three sequential steps. It is contemplated that
each step of this sequential screening will eliminate candidates
from further development. These steps include:
[0218] 1) Identification of compounds which inhibit a microbial
ketol-isomerase activity in vitro;
[0219] 2) Identification of those compounds identified in the first
step which preferentially inhibit a microbial ketol-isomerase
compared to a subject's (e.g., a mammalian) ketol-isomerase in
vitro; and
[0220] 3) Identification of compounds from the second step which
have antimicrobial activity.
[0221] Optionally, candidate compounds may also be tested in an in
vivo antimicrobial system, such as an in vivo mouse
candidosis/candidiasis or aspergillosis model system and/or an in
vivo bacterial infection model system. In some embodiments, these
enzymatic assays, colorimetric quantitations, and antimicrobial
assays described herein are automated to facilitate high throughput
screening (e.g., the Biomek 2000 robot from Beckman Instruments,
Inc.)
[0222] The present invention finds use in the identification of
compounds (i.e., drugs) that have therapeutic value in eliminating,
mitigating and/or preventing microbial infections and/or disease.
Examples of conditions, diseases and disorders which can be treated
using compounds identified using the methods of the present
invention are listed herein. However, it is not intended that the
present invention be limited to treating only those conditions,
diseases and disorders listed herein, as the present invention
finds use in the treatment of additional diseases, disorders or
conditions. Similarly, it is not intended that the present
invention be limited to the treatment of only those diseases,
disorders or conditions caused by microbial species listed herein.
Rather, the present invention also finds use in treating microbial
pathology caused by organisms other than those specifically recited
herein.
[0223] The present invention solves a need in the art for methods
to identify new classes of compounds which have antimicrobial
therapeutic activity against various pathogenic and opportunistic
fungi and bacteria.
EXPERIMENTAL
[0224] The following Examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
[0225] As used herein, the following scientific
abbreviations/notations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); N (Normal); mol (moles); mmol (millimoles); .mu.mol
(micromoles); nmol (nanomoles); g (grams); mg (milligrams); .mu.g
(micrograms); ng (nanograms); l or L (liters); mL (milliliters);
.mu.L (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); MW (molecular weight); .degree. C.
(degrees Centigrade); OD (optical density); EDTA
(ethylenediamine-tetracetic acid); SDS (sodium dodecyl sulfate);
PAGE; UV (ultraviolet); .mu.g/mL (microgram per milliliter); mm
(millimeter); .times.g (times gravity); HPLC (high pressure liquid
chromatography); DDT (dithiothreitol); PMSF (phenylmethylsulfonyl
fluoride); SDS (sodium dodecyl sulfate); s and sec (seconds), m and
min (minutes), h and hr (hours), w/v (weight to volume measure),
v/v (volume to volume measure).
[0226] Restriction enzymes, other common molecular biology enzymes
and reagents used in these experiments are widely available from
numerous sources, including NEB, Promega, Fisher and Stratagene.
Fungal and bacterial growth media (e.g., YG and LB, and
agar-containing formulations of these media) are well known in the
art and are available from suppliers such as Difco.
[0227] As used herein, the following abbreviations apply: ATCC
(American Type Culture Collection, Manassas, Va.); Beckman
Instruments, Inc. (Beckman Instruments, Inc., Fullerton, Calif.);
Charles River Laboratories (Charles River Laboratories, Inc.,
Wilmington, Mass.); Clonetech (Clonetech, Palo Alto Calif.); DIFCO
or Difco (Difco Laboratories, Detroit, Mich.); Fisher (Fisher
Scientific, Pittsburgh, Pa.); Gibco-BRL or BRL or Life Technologies
(GIBCO BRL Life Technologies, Gaithersburg, Md.); ICN (ICN
Biochemicals, Inc., Costa Mesa, Calif.); Molecular Devices
(Molecular Devices Corp., Sunnyvale, Calif.); Promega (Promega
Corp., Madison, Wis.); Sigma (Sigma Chemical Company/Aldrich, St.
Louis, Mo.); Stratagene (Stratagene, La Jolla, Calif.); and Whatman
(Whatman, Inc., Clifton, N.J.).
EXAMPLE 1
Determination of Glutamate Concentration by the Nitro BT Assay
[0228] In this Example, experiments to determine the relationship
between glutamate concentration and the reduced form of nitro BT
using the nitro BT colorimetric assay of the present invention
(See, FIG. 2) are described. In preliminary experiments, glutamine
standard solutions of known concentration were tested.
[0229] Within the wells of a 96 well microtiter plate, 50 .mu.L
aliquots of glutamate standard solution (0, 20, 40, 60, 80, 100,
200, 400 or 600 .mu.M glutamate) were combined with 50 .mu.L of
nitro BT reaction buffer (2 mM nicotinamide adenine dinucleotide,
oxidized form [NAD; Sigma], 16 .mu.M phenazine methosulfate [PMS;
Sigma], 1 mM nitro blue tetrazolium chloride [NBT or nitro BT;
Sigma, Catalog No. N-6876], and 5 units/mL glutamate dehydrogenase
[Sigma, Catalog No. G-2501] in 100 mM potassium phosphate buffer
(KH.sub.2PO.sub.4 and K.sub.2HPO.sub.4, pH 7.8). The mixture was
incubated for 15 min at room temperature, followed by
spectrophotometric (optical density, OD) analysis at 585 nm using a
SPECTRAmax.RTM. 340 microplate reader (Molecular Devices
Corp.).
[0230] The results of this experiment are shown in FIG. 3, in which
the plot depicts OD.sub.585 nm as a function of glutamate standard
concentration, with glutamate concentrations ranging from 0 to 600
.mu.M. Each data point indicates the mean OD.sub.585 nm of
triplicate samples.+-.the standard deviation (S.D.). As can be seen
in the Figure, glutamate concentrations within this range yielded a
linear response with respect to the colorimetric detection (i.e.,
reduction) of nitro BT.
EXAMPLE 2
Preparation of Aspergillus fumigatus Crude Cell Lysate
[0231] In this Example, the growth, harvesting and preparation of
Aspergillus fumigatus crude cell lysates are described. The
resulting crude cell extract is used in the ketol-isomerase
reaction, as described in Examples 3 and 6.
[0232] A. fumigatus ATCC16424 (a human clinical isolate) was
inoculated into liquid YG medium (1% yeast extract [w/v] and 1%
glucose [w/v]) with a final inoculum concentration of
1.times.10.sup.7 conidia per/mL, and incubated for 20 hours at
37.degree. C. with shaking (250 rpm). Hyphae were harvested by
vacuum filtration over Whatman No.2 filter paper, washed twice with
ice-cold water, quick-frozen in dry ice, and stored at -80.degree.
C. until use.
[0233] Frozen A. fumigatus hyphae were thawed and disrupted by
bead-beating (6.times.30 seconds pulses with 2 minutes cooling
between each pulse) using 0.5 mm zirconium beads in KI buffer (600
mM sucrose, 1 mM KCl, 1 mM EDTA and 50 mM PIPES, pH 6.8). The
lysates were centrifuged at 1,000.times.g for 10 minutes at
4.degree. C. The supernatants were recovered and stored at
-80.degree. C. and used as the source material for the
ketol-isomerase assay.
EXAMPLE 3
Ketol-Isomerase Reaction
[0234] In this Example, a source of fungal ketol-isomerase activity
was used to catalyze the formation of glucosamine-6-phosphate
(GlcN-6-P) and glutamate (Glu) from fructose-6-phosphate (Fru-6-P)
and glutamine (Gln). The end products of this in vitro reaction
were assayed using the quantitation methods described in Example 4
(i.e., the Morgan-Elson assay) and Example 1 (i.e., the nitro BT
assay). A direct comparison of these two methods was then made
(See, Example 5 and FIG. 4).
[0235] The ketol-isomerase enzymatic reaction was conducted in a
total final volume of 100 .mu.L, which contained 10 mM Fru-6-P
(Sigma), 10 mM Gln (ICN) and 0.3 mg protein/mL of crude Aspergillus
fumigatus (ATCC No. 16424) cell extract prepared as described in
Example 2. The reaction was incubated at 37.degree. C. for 0, 20,
40 or 60 minutes. The reaction was terminated by boiling for 5
minutes, followed by centrifugation at 10,000.times.g. The
supernatant was recovered and used in the Morgan-Elson and nitro BT
quantitation assays. The ketol isomerase reaction, as well as the
quantitative Morgan-Elson and nitro BT assays were performed in
triplicate to ensure reproducibility.
EXAMPLE 4
Morgan-Elson Assay
[0236] In this Example, a modified Morgan-Elson assay used to
quantitate the concentration of glucosamine-6-phosphate (GlcN-6-P)
in a sample following the ketol-isomerase reaction (described in
Example 3) is described. Thus, this method was used to quantitate
ketol-isomerase activity in the A. fumigatus cell extract.
[0237] A modified Morgan-Elson assay was used to determine GluN-6-P
concentration (Daniels et al., J. Clin. Invest., 97:1235-1241
[1996]). Briefly, 5 .mu.L of saturated NaHCO.sub.3 solution and 5
.mu.L of cold 5% acetic acid were combined with 30 .mu.l of the
ketol-isomerase reaction supernatant, as described in Example 3.
The mixture was incubated at room temperature for 3 minutes, then
boiled for 3 minutes, followed by the addition of 50 .mu.L of 0.27
M sodium borate. Following addition of the sodium borate solution,
the tubes were boiled again for 8 minutes, then cooled to room
temperature. Then, 400 .mu.L of Ehrlich's reagent (1.0 g
p-dimethylaminobenzaldehyde dissolved in 1.25 mL 10 N HCl, then
diluted to 100 mL with glacial acetic acid) was added, followed by
incubation at 37.degree. C. for 20 minutes and colorimetric
analysis (OD.sub.585 nm). Color development is directly
proportional to the generation of GluN-6-P from the ketol-isomerase
assay. Samples of GluN-6-P of known concentration were analyzed in
parallel as standardization values to determine absolute GluN-6-P
concentration in the experimental samples.
EXAMPLE 5
Direct Comparison of Morgan-Elson and Nitro BT Assays
[0238] In this Example, the modified Morgan-Elson and nitro BT
assays were used to quantitate the concentrations of
glucosamine-6-phosphate (GlcN-6-P) and glutamate, respectively, in
a sample. In particular, this comparison was made after the
ketol-isomerase reaction described in Example 3 was conducted. The
Morgan-Elson and the nitro BT assays were conducted according to
the protocols described in Examples 4 and 1, respectively. Thus,
ketol-isomerase activity in the A. fumigatus cell extract was
determined using both methods, and the results of these two assays
were compared. This experiment tested the usefulness (i.e.,
reliability) of the nitro BT assay to quantitate ketol-isomerase
activity, using the Morgan-Elson assay as a comparison.
[0239] The Morgan-Elson assay was conducted as described in Example
4. The nitro BT assay was performed in the wells of a 96 well
microtiter plate, where 50 .mu.L of the ketol-isomerase reaction
mixture was combined with 50 .mu.L of the nitro BT reaction buffer
(pH 8.2), incubated at 37.degree. C. for 90 minutes, followed by
the quantitation of color development at OD.sub.585 nm.
[0240] The results of this side-by-side comparison are shown in
FIG. 4. As can be seen in this Figure, there were no discrepancies
between the stoichiometric quantities of glutamate and GlcN-6-P
following the ketol-isomerase reaction with A. fumigatus cell
extract, as determined by the nitro BT and the Morgan-Elson assays,
respectively. In a control ketol-isomerase reaction, the substrate
glutamine was omitted. In that control reaction, neither glutamate
nor GlcN-6-P were detected (data not shown). From these results, it
is apparent that the nitro BT assay is able to quantitate
ketol-isomerase activity at least as effectively as the
Morgan-Elson assay.
EXAMPLE 6
Detection of Inhibitors of Ketol-Isomerase by the Nitro BT
Assay
[0241] In this Example, experiments conducted to determine whether
the nitro BT colorimetric assay is able to detect whether a
compound has the ability to suppress the ketol-isomerase activity
from an A. fumigatus cell extract. In this Example, the compound
being tested was UDP-N-acetylglucosamine (UDP-GlcNAc), which is
known to cause feedback inhibition of Neurospora crassa
ketol-isomerase (Endo et al., J. Bacteriol., 103:588-594 [1970]).
The UDP-GlcNAc was tested at a concentration range from 0 to 10
mM.
[0242] In this experiment, 50 .mu.L aliquots of a ketol-isomerase
reaction mixture containing 10 mM Fru-6-P, 10 mM Gln, 0.3 mg/mL
crude A. fumigatus extract, and various concentrations of
UDP-GlcNAc were incubated at 37.degree. C. for 60 minutes. The
reaction was terminated by heating at 70.degree. C. for 15 min,
followed by addition of 50 .mu.L of nitro BT reaction buffer, pH
8.2 (See, Example 1). After incubation at 37.degree. C. for 90
minutes, the color development was quantitated by OD.sub.585
nm.
[0243] The results of this assay are shown in a bar-graph format in
FIG. 5. Each data point is the mean of three samples; the standard
deviation of each data point is indicated by vertical lines. As
indicated in the Figure, the ketol-isomerase activity contained in
the A. fumigatus crude extract was inhibited by UDP-GlcNAc in a
dose-dependent manner. The IC.sub.50 (the concentration of compound
which inhibits enzymatic activity by 50% relative to an untreated
control) was 0.48 mM. This inhibitory effect is similar to that
previously reported for UDP-GlcNAc to inhibit ketol-isomerase
activity in crude cell extract of Candida albicans ATCC10261
yeast-like cells (IC.sub.50=0.69 mM; Milewski et al., J. Biol.
Chem., 274:4000-4008 [1999]). These results indicate that
ketol-isomerase inhibitors can be successfully detected using the
nitro BT assay of the present invention.
EXAMPLE 7
Assays for Identifying Compounds Having Antibacterial Activity
[0244] In this Example, protocols are provided for the assay of
antibacterial activity. These assays can be used to screen
compounds previously identified as having
ketol-isomerase-inhibiting activity, to determine whether these
compounds also contain antibacterial activity. Such antibacterial
assays are common in the art, and it is not intended that the
present invention be limited to those assays described herein.
[0245] Agar diffusion and microbroth dilution antibacterial
activity assays are provided. The microbroth dilution method can be
used in preliminary examination, as the method is more readily
adaptable for large-scale screening. The microbroth dilution method
is also suitable for use when a quantitative result is desired, as
this method permits the quantitation of antibacterial activity by
the determination of a "minimum inhibitory concentration" (i.e.,
MIC). The incubation periods, media and temperatures recited in
these methods are intended to be exemplary only, as other bacterial
growth conditions, as known in the art, also find use with the
present invention.
[0246] Agar Diffusion Method--Muller-Hinton agar medium (Difco) is
autoclaved and allowed to cool to 40-50.degree. C. While the medium
is still molten, a bacterial suspension (e.g., S. aureus, E. coli
or P. aeruginosa) is inoculated into the medium and mixed to give a
final concentration of 10.sup.5 -10.sup.6 cells/mL. A suitable
volume of the still-molten mixture is poured into culture dishes on
a level surface to give a uniform agar depth of about 4 mm, and
allowed to cool to room temperature. Two .mu.L of a test solution
containing the candidate antibacterial compound is placed onto the
agar surface. Alternatively, absorbent disks containing known
amounts of the candidate test antimicrobials are placed on the agar
surface. The plates are incubated at 35-37.degree. C. for 16-24
hours. Antibacterial activity is determined by measuring the
diameters (i.e., zones) of growth inhibition.
[0247] Microbroth Dilution Method--Serial dilutions of a solution
containing a candidate antibacterial compound are made in 100 .mu.L
of Muller-Hinton liquid broth (Difco) in the wells of a 96-well
microtiter plate. An additional 5 .mu.L of physiological saline
containing ca. 10.sup.7 cells/mL of a test bacterial strain (e.g.,
S. aureus, E. coli or P. aeruginosa) are then added to the wells.
Plates are incubated at 35-37.degree. C. for 16-24 hours. The
amount of growth in each well is determined either by eye or by
using an automated microplate reader capable of measuring turbidity
and/or light absorbance.
EXAMPLE 8
Assays for Identifying Compounds Having Antifungal Activity
[0248] In this Example, protocols are provided for the assay of
antifungal activity. These assays can be used to screen compounds
previously identified as having ketol-isomerase-inhibiting
activity, to determine whether these compounds also contain
antifungal activity. Such antifungal assays are common in the art,
and it is not intended that the present invention be limited to the
assays described herein.
[0249] Agar diffusion and microbroth dilution antifungal activity
assays are provided in this Example. The microbroth dilution method
can be used in preliminary examination, as the method is more
readily adaptable for large-scale screening. The microbroth
dilution method is also suitable for use when a quantitative result
is desired, as this method permits the quantitation of antifungal
activity by the determination of a "minimum inhibitory
concentration" (i.e., MIC). The incubation periods, media and
temperatures recited in these methods are intended to be exemplary
only, as other fungal growth conditions, as known in the art, also
find use with the present invention.
[0250] Agar Diffusion Method--RPMI medium (Life Technologies)
containing 1.5% agar is autoclaved and allowed to cool to
40-50.degree. C. While the medium is still molten, a fungal
suspension (e.g., A. fumigatus or C. albicans) is inoculated into
the medium and mixed to give a final concentration of 10.sup.6
cells/mL. From this still-molten mixture, a suitable volume is
poured into culture dishes on a level surface to give a uniform
agar depth of about 4 mm, and allowed to cool to room temperature.
Two .mu.L of a test solution containing the candidate antibacterial
compound is placed onto the agar surface. Alternatively, absorbent
disks containing known concentrations of the candidate test
antimicrobials are placed on the agar surface. The plates are
incubated for 18-24 hours at 35.degree. C. (C. albicans) or
37.degree. C. (A. fumigatus). Antifungal activity is determined by
measuring the diameters (i.e., zones) of growth inhibition in the
agar.
[0251] Microbroth Dilution Method--Serial dilutions of a candidate
antifungal compound are made in 100 .mu.L of RPMI medium (Life
Technologies) in the wells of a 96-well microtiter plate. An
additional 100 .mu.L RPMI medium containing 2.times.10.sup.4
conidia/ml of a test fungal strain is then added to these wells.
Conidia from C. albicans, A. fumigatus or any other suitable fungal
species, are suitable for use in this assay. Plates are incubated
for 48 hours with gentle shaking at 37.degree. C. The amount of
growth in each well is estimated either by eye, by microscope
examination, or by using an automated 96-well microplate reader
capable of measuring turbidity and/or light absorbance. In some
embodiments, the methods are adapted so as to allow high throughput
screening of large numbers of compounds. In additional embodiments,
robotic automation is also used.
[0252] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in enzymology, medical
microbiology or related fields are intended to be within the scope
of the following claims.
* * * * *
References